Method and device for synchronously separating liquid phase conformation and gas phase conformation and application

Abstract

The invention discloses a method and device for synchronously separating liquid phase conformation and gas phase conformation and application. The method for synchronously separating the liquid-phase conformation and the gas-phase conformation comprises the step of carrying out online synchronous separation on the liquid-phase conformation and the gas-phase conformation of an analyte in a non-denatured state in a non-denatured solution environment. According to the method and the device for synchronously separating the liquid-phase conformation and the gas-phase conformation, the non-denaturing structure of the analyte is retained by adopting the non-denaturing solution, so that different liquid-phase conformations can be separated by adopting the liquid-phase migration time difference, and meanwhile, different gas-phase conformations are separated by adopting the gas-phase drift time difference; and separation, analysis and correlation analysis of gas-phase conformation and liquid-phase conformation of an analyte in a non-denatured state are realized.

Description

Technical Field

[0001] The present application relates to the technical field of liquid phase conformation and gas phase conformation correlation analysis, and in particular to a method, device and application for synchronously separating liquid phase conformation and gas phase conformation. Background Art

[0002] Ion mobility (IM) technology can separate protein ions of different charges, sizes and shapes in a mobility cell through inert gas collision and electric field induction, and measure the collision cross section (CCS) of proteins in the gas phase; combining it with mass spectrometry (MS) that can accurately measure the molecular weight of proteins, namely IM-MS, can be applied to study the conformational differences and conformational changes of proteins. However, the analysis object of IM-MS is essentially protein ions in the gas phase, and their conformation may be different from the original conformation of the protein in solution.

[0003] Capillary electrophoresis (CE) has the ability to separate protein conformations in a non-denaturing solution environment. However, due to the complexity of electrophoretic migration in solution, it is difficult to directly obtain the size information of protein analytes based on CE migration.

[0004] If CE and IM (including CE-IM-MS) can be combined, the coexisting conformations of proteins can be separated and characterized in the solution phase and gas phase respectively, and the correlation between the two-phase conformations can be verified, thereby improving the accuracy of conformational characterization. Since protein conformations can change dynamically and the CE flow rate is extremely low, making it difficult to collect fractions for offline IM analysis, CE-IM needs to be used in conjunction online. There are currently literature reports on the combined use of CE-IM-MS technology, which is mostly used for multidimensional separation and analysis of smaller molecules, such as high-sensitivity detection of small molecules and separation and analysis of peptides. Although CE-IM-MS methods have been used in protein analysis, such as top-down dissociation analysis of proteins, data are mostly collected under denaturing conditions, and therefore it is impossible to obtain non-denaturing conformational information of proteins. Summary of the invention

[0005] The purpose of the present application is to provide an improved method, device and application for synchronously separating liquid phase conformation and gas phase conformation.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions:

[0007] One aspect of the present application discloses a method for synchronously separating liquid phase conformations and gas phase conformations, which is characterized by: comprising online synchronous separation of liquid phase conformations and gas phase conformations of an analyte in a non-denaturing solution environment.

[0008] In this application, "online" means that the liquid phase effluent is sampled into the gas phase separation device in real time, that is, the analyte enters the gas phase separation in real time after the liquid phase separation, and the two are carried out continuously in one detection process. "Synchronous" means that it is completed in the same detection process, without the need for two experiments, and the liquid phase conformation and the gas phase conformation are correlated, and there is no time sequence relationship.

[0009] It should be noted that the method of the present application can separate the gas phase conformation and liquid phase conformation of the analyte in a non-denaturing state under non-denaturing conditions, laying the foundation for subsequent analysis or correlation analysis of the gas phase conformation and liquid phase conformation of the analyte in a non-denaturing state.

[0010] In one implementation of the present application, liquid phase separation is performed based on the difference in liquid phase migration time of analytes in different conformations.

[0011] In one implementation of the present application, gas phase separation is performed based on the difference in gas phase drift time of analytes in different conformations.

[0012] In one implementation of the present application, liquid phase separation includes capillary electrophoresis separation.

[0013] In one implementation of the present application, gas phase separation includes ion mobility or differential mobility analysis.

[0014] In one implementation of the present application, the liquid phase conformation and the gas phase conformation of the same analyte in a non-denatured state are separated online synchronously.

[0015] In one implementation of the present application, the analyte includes natural molecules or synthetic molecules that have the same molecular formula but have different conformations or can undergo conformational interconversion.

[0016] In one implementation of the present application, the analyte includes at least one of proteins, nucleic acids, artificial synthetic polymers, polysaccharides and small organic molecules, or a complex formed by at least one of proteins, nucleic acids, artificial synthetic polymers, polysaccharides and small organic molecules.

[0017] It should be noted that the complex of the present application can be a complex formed by molecules of the same type, such as a complex formed by two or more proteins, or a complex formed by molecules of different types, such as a complex formed by protein and nucleic acid.

[0018] In one implementation of the present application, molecules of the same or different types in the complex are bound to each other through non-covalent interactions.

[0019] One implementation of the present application includes using a combination of liquid phase separation and gas phase separation technology to synchronously separate the liquid phase conformation and gas phase conformation of the analyte in a non-denaturing state in a non-denaturing solution environment.

[0020] Another aspect of the present application discloses a method for synchronously analyzing liquid phase conformation and gas phase conformation, including collecting the method for synchronously separating liquid phase conformation and gas phase conformation of the present application to separate and obtain liquid phase conformation data and gas phase conformation data of the analyte, and performing online synchronous analysis of the liquid phase conformation data and the gas phase conformation data.

[0021] In one implementation of the present application, the liquid phase conformational data includes liquid phase migration time (MT), mass-to-charge ratio (m / z) of the analyte, and signal intensity (intensity).

[0022] In one implementation of the present application, the gas phase conformational data includes gas phase drift time (DT), mass-to-charge ratio of the analyte, and signal intensity.

[0023] In one implementation of the present application, the collected liquid phase conformation data and gas phase conformation data are processed to obtain a data graph.

[0024] In one implementation of the present application, the collected liquid phase conformation data is processed to obtain the liquid phase conformation information of the analyte.

[0025] In one implementation of the present application, the collected gas phase conformation data is processed to obtain the gas phase conformation information of the analyte.

[0026] Another aspect of the present application discloses a method for synchronously analyzing the correlation between liquid phase conformation and gas phase conformation, comprising collecting the liquid phase conformation data and gas phase conformation data of the analyte by the method of synchronously separating liquid phase conformation and gas phase conformation of the present application, and performing correlation analysis on the liquid phase conformation data and the gas phase conformation data.

[0027] In one implementation of the present application, the correlation analysis includes performing a correlation analysis between the gas phase conformation and the liquid phase conformation of the analyte in a non-denatured state based on the correlation between the gas phase separation and liquid phase separation behaviors of the analyte, and the correlation between the liquid phase migration time and the gas phase drift time.

[0028] In one implementation of the present application, taking protein analytes as an example, four-dimensional data information of protein analytes is collected to perform correlation analysis between gas phase conformation and liquid phase conformation; wherein the four-dimensional data information includes mass-to-charge ratio, signal intensity, gas phase drift time, and liquid phase migration time. Specifically, the collected four-dimensional data information is processed to obtain protein molecular weight information of the protein analyte, an MT diagram representing protein liquid phase structure information, a collision cross-sectional area diagram representing protein gas phase structure information, a DT-intensity diagram under a specified m / z-MT, and an MT-intensity diagram under a specified m / z-DT.

[0029] In the method for synchronous separation of liquid phase conformation and gas phase conformation, the synchronous analysis method and the synchronous analysis correlation method of the present application, online synchronous separation uses a combination of a liquid phase separation device and a gas phase separation device.

[0030] In one implementation of the present application, the liquid phase separation device is a capillary electrophoresis instrument or an instrument with similar functions.

[0031] In one implementation of the present application, the gas phase separation device is an ion mobility meter, a mass spectrometer containing an ion mobility module, a differential mobility analyzer, or an instrument with similar functions.

[0032] In one implementation of the present application, a capillary electrophoresis instrument is connected to an interface device equipped with a capillary glass spray needle, and a positive voltage is connected to the capillary injection end of the capillary electrophoresis instrument to provide an electric field required to drive the solution to move. A negative voltage is provided by a gas phase analysis device, and the negative voltage is connected to the solution in the interface device, so that the injection end of the capillary electrophoresis instrument, the capillary, the solution in the interface device and the electrospray form a stable electrical circuit; the capillary electrophoresis instrument is used to perform liquid phase separation and detection on the analyte, and then the analyte enters the interface device, forms an electrospray through the capillary glass spray needle, and enters the gas phase separation device for gas phase separation and detection.

[0033] In one implementation of the present application, the interface device includes an interface device body, which includes at least a first interface, a second interface and a third interface; the first interface is used to connect to the outlet end of the liquid phase separation device; the second interface is used to connect to the capillary glass spray needle to receive the analyte exported from the outlet end of the liquid phase separation device, and send the analyte into the gas phase separation device in the form of a spray through the capillary glass spray needle; the third interface is used to connect a non-denaturing solution, and when in use, the third interface is connected to a negative voltage to form a spray voltage circuit.

[0034] In one implementation of the present application, the inner diameter of the outlet end of the capillary glass spray needle is in the micron level.

[0035] In one implementation of the present application, the inner diameter of the capillary glass spray needle body is 150 μm-1 mm.

[0036] It should be noted that the present application uses a glass capillary with a main body inner diameter of 150μm-1mm, and uses a needle puller to pull out a tip at its outlet end, that is, to obtain a capillary glass spray needle with an outlet inner diameter of micrometer level. It can be understood that the tip pulled by the needle puller has a smaller inner diameter than the spray needle body, that is, the inner diameter of the outlet end is smaller than the inner diameter of the spray needle body.

[0037] It should also be noted that the present application uses a capillary glass spray needle with an inner diameter of micrometers at the outlet end, which can improve the ionization efficiency, so that the analyte in the non-denaturing solution can also be ionized, separated and detected by the gas phase separation device, thereby obtaining the gas phase conformation information of the non-denaturing state of the analyte under non-denaturing conditions.

[0038] In one implementation of the present application, the third interface is disposed below and to the side of the interface between the second joint and the capillary glass spray needle.

[0039] It should be noted that in a further improvement scheme of the present application, the third interface is arranged at the lower side of the interface between the second joint and the capillary glass spray needle, which can ensure that the non-denaturing solution, especially the static solution, is more stably and continuously input into the capillary glass spray needle, avoiding the problem of unstable spray caused by high vacuum and rapid solution consumption, thereby further improving the spray stability.

[0040] In one implementation of the present application, the interface device body also includes a fourth interface for connecting the auxiliary modification liquid. When in use, the third interface is not connected to the negative voltage, and the fourth interface is connected to the negative voltage to form a spray voltage circuit.

[0041] In one implementation of the present application, the fourth interface is disposed below and to the side of the interface between the second joint and the capillary glass spray needle.

[0042] It should be noted that the fourth interface is arranged below the interface between the second joint and the capillary glass spray needle, also to ensure that the auxiliary modification liquid can be stably and continuously input into the capillary glass spray needle, thereby improving the spray stability.

[0043] In one implementation of the present application, the interface device further includes an interface support frame for supporting and installing the interface device body.

[0044] In one implementation of the present application, an ion source identification contact is provided on the interface support frame for activating the mass spectrometer.

[0045] It should be noted that the ion source identification contacts on the interface support frame of the present application, that is, the identification contacts that come with the mass spectrometer, are removed together with the spray needle device that comes with the mass spectrometer in the present application, and installed on the interface support frame of the present application so that it is located near the ion source. The mass spectrometer determines whether the ion source is correctly installed by whether the ion source identification contacts are conductive. If it is determined that no ion source is installed, signal acquisition cannot be started; the identification contacts on the interface support frame are used to provide the mass spectrometer with a judgment on how to start signal acquisition.

[0046] In one implementation of the present application, the non-denaturing solution is a non-denaturing sheath fluid.

[0047] In one implementation of the present application, the non-denaturing solution is a static sheath solution or a continuous flow sheath solution.

[0048] Another aspect of the present application discloses a combined instrument, including a liquid phase separation device and a gas phase separation device; the liquid phase separation device is connected to an interface device equipped with a capillary glass spray needle, the injection end of the liquid phase separation device is connected to a positive voltage to provide an electric field required to drive the solution to move, and the negative voltage is provided by the gas phase separation device, and the negative voltage is connected to the solution in the interface device, so that the injection end of the liquid phase separation device, the solution in the interface device and the electrospray form a stable electrical circuit; the interface device is connected to a non-denaturing solution.

[0049] It should be noted that the combined instrument of the present application is actually a combined instrument using a liquid phase separation device and a gas phase separation device for online synchronous separation in the method for synchronously separating liquid phase conformation and gas phase conformation of the present application; therefore, its specific definition can refer to the method for synchronously analyzing liquid phase conformation and gas phase conformation of protein of the present application, for example, the electrophoresis device is a capillary electrophoresis instrument, the gas phase analysis device is an ion mobility instrument, a mass spectrometer containing an ion mobility module, or a differential mobility analyzer, the interface device includes an interface device body, the interface device body is a three-way interface or a four-way interface, the interface device can also include an interface support frame or an interface support frame with ion source identification contacts, a capillary glass spray needle with an inner diameter of micrometers at the outlet end, etc., which are not repeated here.

[0050] Another aspect of the present application discloses the application of the combined instrument of the present application in 1) synchronous separation of liquid phase conformation and gas phase conformation, 2) synchronous analysis of liquid phase conformation and gas phase conformation or 3) synchronous analysis of the correlation between liquid phase conformation and gas phase conformation.

[0051] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0052] The present invention discloses a method and device for synchronously separating liquid phase conformations and gas phase conformations, which uses a non-denaturing solution to retain the non-denaturing structure of the analyte, so that different liquid phase conformations can be separated by using differences in liquid phase migration time. At the same time, different gas phase conformations can be separated by using differences in gas phase drift time, thereby achieving separation, analysis and correlation analysis of the gas phase conformation and liquid phase conformation of the analyte in a non-denaturing state. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is a top cross-sectional view of the interface device body in the embodiment of the present application;

[0054] Figure 2 is a positional relationship diagram of the interface device body and the interface support frame in the embodiment of the present application;

[0055] Figure 3 This is a schematic diagram of the CE-IM-MS technology in the embodiments of the present application;

[0056] Figure 4is the mass spectrum of three ion types of NB28 in the embodiment of the present application;

[0057] Figure 5 MT diagram and CCS diagram of three ion types of NB28 in the embodiment of the present application;

[0058] Figure 6 These are the MT diagram and DT diagram of the NB28Ⅲ ion type in the examples of this application. DETAILED DESCRIPTION

[0059] In order to compare the solution phase and gas phase structures of proteins, the present application has developed a non-denaturing capillary electrophoresis-ion mobility-mass spectrometry (CE-IM-MS). On the one hand, the separation mechanism of capillary electrophoresis is based on the electrophoretic mobility of proteins in the liquid phase, which can be adjusted according to the selection of background electrolyte (BGE) and coating. On the other hand, the drift time of ions in ion mobility mass spectrometry is related to the gas phase mobility of ions. Therefore, the combination of these two techniques can analyze the solution phase and gas phase conformation of the same protein, which can provide multi-dimensional information for the study of protein conformation and conformational changes.

[0060] It is understood that the above combination scheme is not only applicable to proteins, but also to other similar natural or synthetic molecules with the same molecular formula but different conformations or conformational interconversion, including but not limited to proteins, nucleic acids, synthetic polymers, polysaccharides, small organic molecules, or complexes of the same or different molecules of these substances. Therefore, the method of synchronously separating liquid phase conformation and gas phase conformation of the present application includes online synchronous separation of liquid phase conformation and gas phase conformation of the analyte in a non-denaturing solution environment.

[0061] It should be noted that the method of the present application performs online synchronous separation of liquid phase conformation and gas phase conformation in a non-denaturing environment. On this basis, it can further realize online synchronous analysis of liquid phase conformation and gas phase conformation and correlation analysis between the two, providing a new solution and approach for the analysis and correlation analysis of liquid phase conformation and gas phase conformation in a non-denaturing state of the analyte.

[0062] In one implementation of the present application, the non-denaturing solution is a non-denaturing sheath liquid; wherein the sheath liquid forms include static sheath liquid and continuous flow sheath liquid. Static sheath liquid avoids problems such as spray instability caused by flowing sheath liquid and decreased sensitivity due to dilution. The continuous flow sheath liquid injects the capillary electrophoresis sheath liquid into the small inner diameter nanoliter electrospray needle of the capillary electrophoresis-ion mobility mass spectrometry interface in the form of a low flow rate continuous flow. The small inner diameter of the electrospray needle is used to improve the ionization efficiency. This sheath liquid can be used to improve the spray stability under non-denaturing conditions; in short, the low flow rate continuous flow sheath liquid has the advantages of strong flow rate controllability and more stable spray. In addition, the low flow rate continuous flow sheath liquid can avoid changes in solution parameters because it is supplemented with fresh solution. The solution parameters include but are not limited to pH, ionic strength, salt concentration, concentration of other components, etc.; overcome the problem of changes in solution parameters such as pH caused by the accumulation of a single electrolytic electrolyte during the continuous operation of CE in the static sheath liquid, and effectively prevent artificial changes in protein conformation. Therefore, static sheath liquid or low flow rate continuous flow sheath liquid can be selectively used according to experimental requirements. The flow rate of the low-flow continuous flow sheath fluid is lower than 5 μL / min, preferably lower than 2 μL / min. In a more preferred embodiment, the flow rate of the low-flow continuous flow sheath fluid does not exceed 1 μL / min.

[0063] The present application is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other devices, materials, and methods. In some cases, some operations related to the present application are not shown or described in the specification in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and the related operations can be fully understood according to the description in the specification and the general technical knowledge in the art.

[0064] Example

[0065] In order to realize the combined analysis of CE and IM or IM-MS, including the analysis of intact proteins under non-denaturing conditions, a new interface device was first developed in this example.

[0066] The interface device in this example can realize the online coupling of CE and IM or IM-MS. CE is used for the separation and characterization of different conformations of proteins in the solution phase; the interface device itself uses the ESI principle to realize the ionization of intact proteins in the CE outflow solution for subsequent IM or IM-MS analysis; IM is used for the separation and characterization of different conformations of protein ions in the gas phase; MS can be used for the determination of the molecular weight and ion valence of proteins.

[0067] The interface device in this example mainly includes a sheath liquid module and an ionization module. The sheath liquid module includes a static sheath liquid or a continuous flow sheath liquid. The static sheath liquid can avoid the sample dilution problem caused by the continuous flow. The continuous flow sheath liquid is used to provide a stable continuous liquid flow as the sheath liquid of the CE effluent. On the one hand, the total flow rate is increased after merging with the CE effluent to improve the stability of electrospray. On the other hand, it overcomes the pH change problem caused by the accumulation of a single electrolyte in the static sheath liquid pool during the continuous operation of CE in the existing design, and effectively prevents artificial changes in the protein conformation. Therefore, static sheath liquid or continuous flow sheath liquid can be selectively used according to experimental requirements. Because the sample is precious, static sheath liquid is used in this example; the ionization module uses a glass capillary with a fine-caliber tip as a spray needle to ensure efficient ionization of intact proteins at low flow rates.

[0068] The interface device of this example includes an interface device body, such as Figure 1 As shown, the interface device body includes a first interface, a second interface, a third interface, and a fourth interface; wherein the first interface is used to connect to the capillary outlet end of the capillary electrophoresis device; the second interface is used to connect to the capillary glass spray needle to receive the analyte derived from the capillary outlet end of the capillary electrophoresis device, and to deliver the analyte into the mass spectrometer in the form of a spray through the capillary glass spray needle; the third interface is arranged at the lower side of the interface between the second interface and the capillary glass spray needle, and is used to connect the static sheath liquid; the fourth interface is arranged at the lower side of the interface between the second interface and the capillary glass spray needle, and is used to connect the auxiliary modification liquid, and is connected to a negative voltage when in use to form a spray voltage loop.

[0069] The fourth interface for connecting the auxiliary modification liquid can be omitted according to the needs, and in this case, the negative pressure can be connected to the third interface.

[0070] For ease of use, the interface device of this example also includes a number of matching threaded adapters and sleeves for connecting the connectors to corresponding components.

[0071] Furthermore, in order to facilitate the fixing and installation of the interface device, the interface device of this example also includes an interface support frame, such as Figure 2 As shown, it is mainly used to support and install the interface device body. Further, the interface support frame is provided with ion source identification contacts for activating the mass spectrometer.

[0072] The specific instruments and materials used in this case include:

[0073] Commercial ion mobility instrument and mass spectrometer with ion mobility module. This experiment used an ion mobility quadrupole time-of-flight mass spectrometer (SYNAPT XS Ion Mobility Time-of-Flight Mass Spectrometer, Waters); capillary electrophoresis separation instrument (CESI 8000Plus High Performance Separation-ESI Module, AB Sciex).

[0074] CE capillary coated with hydroxypropyl cellulose (HPC), outer diameter 0.15mm; capillary glass needle outer diameter 1mm, inner diameter 0.75mm; FEP sleeve 1 outer diameter 1.59mm, inner diameter 1.07mm, used to fix the capillary glass needle; FEP sleeve 2 outer diameter 1.59mm, inner diameter 0.18mm, used to fix the CE capillary; PEEK threaded adapter inner diameter 1.6mm; sheath liquid tube 1 outer diameter 1.6mm, inner diameter 1mm, connected to the injection The device can be used to introduce non-denatured dynamic sheath liquid. When it is not connected to the syringe or the syringe end does not apply pressure, it can be used as the introduction end of non-denatured static sheath liquid, so the static and dynamic sheath liquids can be switched at any time; the outer diameter of the sheath liquid tube 2 is 1.6mm, the inner diameter is 0.13mm, and it is used to introduce the sheath liquid into the four-way; the outer diameter of the auxiliary modification liquid tube is 1.6mm, the inner diameter is 0.13mm, and it is used to connect the liquid in the auxiliary modification bottle and the four-way; the second-way hole diameter is 1.6mm; the hole diameter of the four-way connector is 1mm; the modification liquid injection bottle; the electrical wire. Among them, the sheath liquid tube 1, the sheath liquid tube 2, and the auxiliary modification liquid tube are all PEEK tubes.

[0075] The principle of the interface device in this example:

[0076] The sample and background electrolyte solution enter the CE capillary under a certain electric field. This electrophoretic separation driven by the electric field is called mobility, denoted by μ, which is the rate of ion migration under unit field strength. In capillary electrophoresis, the charged substance itself will move relative to the background electrolyte solution due to the electrophoretic effect, thus generating "electrophoretic mobility", denoted by μ. p The relationship between electrophoretic mobility and migration velocity is:

[0077] μ p =v / E (Formula 1)

[0078] where μ p is the mobility, v is the velocity, and E is the electric field strength. In a uniform electric field, the electric field force F exerted on a charged particle is E It can be calculated by the electric field strength E and the charge q it carries:

[0079] F E =qE (Formula 2)

[0080] In addition to the electric field force, charged materials are also subject to the resistance of the environment F. F , the size is:

[0081] F F =6πηrv (Formula 3)

[0082] Where η is the viscosity of the solution, and r is the hydrodynamic radius of the substance. The substance itself is not necessarily a sphere, and hydration or other structural changes may occur in the solution. The equivalent radius calculated by macroscopic fluid dynamics methods by treating the substance as a sphere and taking various external factors into account is the so-called "hydrodynamic radius". When the charged substance reaches equilibrium during electrophoresis, the electric field force F E and resistance F F Equal in size and opposite in direction, so we have

[0083] qE=6πηrv (Formula 4)

[0084] Combining equation 1 with equation 4, we can get:

[0085] μ p =q / (6πηr)

[0086] It can be seen that the electrophoretic mobility of a charged substance is proportional to its own charge and inversely proportional to the solution viscosity and its own size. This allows the separation of substances with different charges and conformations. In addition, since the value of charge q can be positive or negative, the electrophoretic mobility μ p The positive and negative of will also change accordingly, and the physical meaning is that substances with different signs of charge have opposite electrophoretic directions. Therefore, the direction of capillary electrophoresis mobility depends on the positive and negative charge of the analyte in the background electrolyte solution. When the isoelectric point (pI) of the analyte is higher than the pH value of the background electrolyte solution, it is positively charged and the electrophoresis direction is toward the capillary outlet. Under the action of electroosmotic flow, the analyte will generally move toward the capillary outlet.

[0087] The sample solution moved to the outlet is desolvated and ionized and enters the ion mobility cell in gaseous form for gas phase separation. In ion mobility mass spectrometry, ions move due to the voltage applied in the mobility cell. Due to the presence of inert gas in the mobility cell, ions with more compact structures are subject to less resistance and have a shorter movement time, thereby achieving the separation of ions with different structures. CE-IM-MS combined technology combines the above two separation technologies to achieve the separation and detection of gas phase and liquid phase structures at the same time.

[0088] Testing process:

[0089] like Figure 3As shown in the figure, when the background electrolyte solution enters the capillary glass needle from the CE capillary, the modification solution will mix with the analyte and form an electrospray voltage loop at the same time. At this time, different analytes are separated due to the difference in the movement mode in the solution under the action of the electric field in the CE capillary. The separated analytes reach the tip of the capillary glass needle in turn to form an electrospray, and then enter the mobility cell in the form of gas phase after desolvation for further separation. The ion mobility method enables ions of different charges, sizes and shapes to be separated in the mobility cell through inert gas collision and electric field induction, and finally their precise molecular weight is determined by mass spectrometry, ultimately achieving separation and detection of analytes in liquid and gas phase environments.

[0090] Application examples:

[0091] 1. Install the CE-IM-MS instrument: connect the capillary electrophoresis instrument and the ion mobility mass spectrometer through the interface device;

[0092] 2. Injection, separation, and detection: The sample is injected through the pressure at the CE injection end and the voltage difference between the inlet and outlet of the CE capillary. At the same time, liquid phase separation is achieved in the CE capillary and gas phase separation is achieved in the ion mobility cell, and finally enters the mass spectrometer for detection;

[0093] 3. Data collection and analysis: The collected m / z, intensity, DT, and MT four-dimensional data were imported into DriftScopeV2.9 software for analysis to obtain protein molecular weight information, MT diagram representing protein liquid phase structure information, collision cross-sectional area diagram (CCS diagram) representing protein gas phase structure information, and DT-intensity diagram under specific m / z-MT, and finally realize the correlation analysis of protein liquid and gas phase structures.

[0094] Specific examples include:

[0095] Install the interface device body: The interface device body includes a first interface, a second interface, a third interface and a fourth interface, namely a four-way connector 8. Figure 1 As shown, the first interface of the four-way connector 8 is connected to the capillary outlet end of the capillary electrophoresis device. Specifically, the capillary 1 is fixed with the FEP sleeve 12, and then installed to the first interface with the PEEK threaded adapter 4; the capillary glass spray needle 2 is installed at the second interface, and similarly, the capillary glass spray needle 2 is fixed with the FEP sleeve 3 and then installed to the second interface with the PEEK threaded adapter; the third interface is connected to the non-denatured static sheath liquid, and the fourth interface is connected to the auxiliary modification liquid. Specifically in this example, one end of the auxiliary modification liquid tube 9 is inserted into the auxiliary modification liquid injection bottle 10, and the other end is inserted into the fourth interface through the PEEK threaded adapter, and the auxiliary modification liquid injection bottle 10 is inserted with an electric conductor 11 for connecting a negative voltage; the sheath liquid tube 1 5 is connected to the sheath liquid tube 2 7 through a two-way structure 6, and then inserted into the third interface through the PEEK threaded adapter.

[0096] Connect CE and the interface device body: insert the inlet end of the CE capillary into the injection bottle, connect the outlet section to the first joint of the interface body, pass through the cross-way and insert into the capillary glass spray needle at the second joint.

[0097] Install the interface body on the IM-MS instrument: remove the baffle, conductive wire and spray needle device on the static source device of the SynaptXS instrument, place the interface support frame with ion source identification contacts on the static source, and finally fix the interface body on the interface support plate. Device inspection: Use a 200μL syringe to inject the background electrolyte into the interface device along the static sheath liquid end. If there is spray at the tip of the capillary glass spray needle, it means that the device is complete. Install the electrical circuit: The positive voltage is located at the capillary injection end, and the 30kV voltage is turned on during the sample measurement; the negative voltage is provided by the mass spectrometer and connected to the modification liquid connected to the fourth connector through the electrical conductor. The voltage is 1.2-1.8kV. Operation: First, inject the background electrolyte into the interface device from the CE capillary injection end to form a stable electrical circuit, and finally separate and detect the analytes.

[0098] Experimental conditions:

[0099] (1) Reagents

[0100] Nanobody 28 (NB28), NB28 protein was purified and dissolved in 10mM PBS solution at pH=7.4; Ochratoxin A (OTA) was purchased from MedChemExpress (USA), OTA powder was dissolved in methanol solution to prepare 2mM OTA solution; Phosphate Buffered Saline (PBS) was purchased from Themo Fisher (USA). Hydroxypropyl cellulose (HPC), ammonium acetate (NH4Ac, HPLC grade), and methanol (MeOH) were all of chromatographic grade purity and purchased from Sigma-Aldrich (USA).

[0101] (2) Sample processing

[0102] All protein solutions were desalted, and a 3kDa ultrafiltration membrane was selected. A 20mM ammonium acetate solution was used for solution replacement to remove the incompatible volatile salts in the protein sample that were incompatible with mass spectrometry. The steps were as follows: first, a 20mM NH4Ac solution was placed in a 3kDa ultrafiltration tube, and centrifuged at 12000g for 5 minutes to activate the filter membrane; then, the sample and 20mM NH4Ac solution were added, and centrifuged at 12000g for 10 minutes; the filtrate was collected after repeated centrifugation 5-6 times. Finally, the protein concentration after desalting was measured.

[0103] NB28 was desalted and diluted to 10 μM. NB28-OTA was prepared at a protein-ligand molar concentration of 2:1; samples should be prepared before use.

[0104] (3) CE-IM-MS experimental conditions and parameter settings

[0105] The capillary used was a HPC coated tube with a length of 100 cm, the BGE was a 20 mM NH4Ac solution, the injection volume was 2 psi, 10 s, the separation voltage was 30 kV, and the pressure of 1 psi was maintained during the separation process.

[0106] NB28 and NB28-OTA were added to the CE sample bottle, and after capillary separation, they were entered into the mass spectrometer for detection. The main parameter conditions of SynaptXS mass spectrometer were set as follows: Capillary Voltage, 1.2-1.7 kV; Source Temperature, 120°C; Trap Collision Energy, 10 V.

[0107] Experimental data:

[0108] Data analysis method: MassLynx V4.2 software (Waters, USA), DriftScope V2.9 (Waters, USA), CIUSuite 2 (GitHub, USA).

[0109] Mass spectra such as Figure 4 As shown; the MT diagram and collision cross-sectional area diagram (CCS diagram) corresponding to a specific charge state and a specific ion type are shown as Figure 5 As shown; the MT diagram corresponding to a specific charge state is as follows Figure 6 As shown in A, the DT diagram corresponding to a specific charge state is as follows Figure 6 As shown in B(1), the DT diagram corresponding to a specific charge state and a specific MT is as follows Figure 6 B(2) to Figure 6 As shown in B(4).

[0110] Figures 4 to 5In the figure, I represents the NB28 protein ion alone, II represents the NB28 protein ion that is not bound to OTA after adding OTA, and III represents the NB28 protein ion that is bound to OTA after adding OTA. Figure 5 In the figure, the left figure is the MT map corresponding to the three ion types of NB28 protein I, II, and III in the +7 charge state, and the right figure is the CCS map corresponding to NB28 I, II, and III in the +7 charge state, where A and B are two different conformations of NB28 protein. Figure 6 A is the MT map corresponding to the III ion type and +7 charge state of NB28 protein; B(1) is the DT map corresponding to the III ion type and +7 charge state of NB28 protein; B(2) is the DT map corresponding to the III ion type and +7 charge state of NB28 protein, MT=38.6-42min; B(3) is the DT map corresponding to the III ion type and +7 charge state of NB28 protein, MT=42-46min; B(4) is the DT map corresponding to the III ion type and +7 charge state of NB28 protein, MT=38.6-46min. The four-dimensional data association information of m / z, intensity, DT and MT is displayed.

[0111] Figures 4 to 6 The results show that the method and device for synchronously analyzing the liquid phase conformation and gas phase conformation of proteins in this example can simultaneously detect the NB28 protein ions in the liquid phase ( Figure 5 Left figure) and gas phase ( Figure 5 2) Two conformations of NB28 in the liquid phase can be separated by CE. 3) Two conformations of NB28 in the gas phase can be separated by IM. 4) The DT value of the two conformations separated by the gas phase drift process ( Figure 6 B(1)), and the DT values ​​corresponding to the two conformations separated by the liquid phase migration process ( Figure 6 B(2) to Figure 6 B(4)), indicating that the liquid phase and gas phase structures of NB28 protein are related.

[0112] In summary, the method and device for synchronously analyzing the liquid phase conformation and gas phase conformation of a protein in this example can obtain four-dimensional data information of the mass-to-charge ratio, signal intensity, gas phase drift time and liquid phase migration time of the protein sample. Based on this, the correlation between the gas phase separation and liquid phase separation behaviors of the NB28 protein and the correlation between its liquid phase migration time and gas phase drift time can be used to perform a correlation analysis of the gas and liquid phase conformations.

[0113] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A method for synchronously separating liquid phase conformation and gas phase conformation, characterized in that: It includes online synchronous separation of the liquid phase conformation and gas phase conformation of the analyte in a non-denaturing solution environment.

2. The method according to claim 1, characterized in that: Liquid phase separation is performed based on the difference in liquid phase migration time of analytes in different conformations; Optionally, performing gas phase separation based on differences in gas phase drift times of analytes in different conformations; Optionally, the liquid phase separation comprises capillary electrophoresis separation; Optionally, the gas phase separation comprises ion mobility or differential mobility analysis; Optionally, performing online simultaneous separation of the liquid phase conformation and the gas phase conformation of the same analyte in a non-denatured state; Optionally, the analyte comprises a natural molecule or a synthetic molecule having the same molecular formula but having different conformations or capable of undergoing conformational interconversion; Optionally, the analyte includes at least one of proteins, nucleic acids, artificial synthetic polymers, polysaccharides and small organic molecules, or a complex formed by at least one of proteins, nucleic acids, artificial synthetic polymers, polysaccharides and small organic molecules; Optionally, the same or different molecules in the complex are bound to each other by non-covalent interactions; Optionally, the method comprises using a combination of liquid phase separation and gas phase separation technology to synchronously separate the liquid phase conformation and gas phase conformation of the analyte in a non-denaturing state in a non-denaturing solution environment.

3. A method for synchronously analyzing liquid phase conformation and gas phase conformation, characterized in that: The method comprises collecting liquid phase conformation data and gas phase conformation data of the analyte separated and obtained by the method according to any one of claims 1 to 2, and performing online synchronous analysis on the liquid phase conformation data and the gas phase conformation data.

4. The method according to claim 3, characterized in that: The liquid phase conformation data include liquid phase migration time, mass-to-charge ratio of the analyte, and signal intensity; Optionally, the gas phase conformation data includes gas phase drift time, mass-to-charge ratio of the analyte, and signal intensity; Optionally, including processing the collected liquid phase conformation data and gas phase conformation data to obtain a data graph; Optionally, including processing the collected liquid phase conformation data to obtain liquid phase conformation information of the analyte; Optionally, the method includes processing the collected gas phase conformation data to obtain gas phase conformation information of the analyte.

5. A method for synchronously analyzing the correlation between liquid phase conformation and gas phase conformation, characterized in that: The method comprises collecting liquid phase conformation data and gas phase conformation data of the analyte separated and obtained by the method according to any one of claims 1 to 2, and performing correlation analysis on the liquid phase conformation data and the gas phase conformation data.

6. The method according to claim 5, characterized in that: The liquid phase conformation data include liquid phase migration time, mass-to-charge ratio of the analyte, and signal intensity; Optionally, the gas phase conformation data includes gas phase drift time, mass-to-charge ratio of the analyte, and signal intensity; Optionally, the correlation analysis includes performing a correlation analysis of the gas phase conformation and the liquid phase conformation of the analyte in a non-denatured state based on the correlation between the gas phase separation and liquid phase separation behaviors of the analyte and the correlation between its liquid phase migration time and gas phase drift time.

7. The method according to any one of claims 1 to 6, characterized in that: The online synchronous separation adopts a combination of a liquid phase separation device and a gas phase separation device; Optionally, the liquid phase separation device is a capillary electrophoresis instrument or an instrument with similar functions; Optionally, the gas phase separation device is an ion mobility meter, a mass spectrometer containing an ion mobility module, a differential mobility analyzer, or an instrument with similar functions; Optionally, the capillary electrophoresis instrument is connected to an interface device equipped with a capillary glass spray needle, the capillary injection end of the capillary electrophoresis instrument is connected to a positive voltage to provide an electric field required to drive the solution to move, and the negative voltage is provided by the gas phase analysis device, and the negative voltage is connected to the solution in the interface device, so that the injection end of the capillary electrophoresis instrument, the capillary, the solution in the interface device and the electrospray form a stable electrical circuit; The analyte is separated and detected in liquid phase by a capillary electrophoresis instrument, and then the analyte enters the interface device, forms an electrospray through the capillary glass spray needle, and enters the gas phase separation device for gas phase separation and detection; Optionally, the interface device comprises an interface device body, and the interface device body comprises at least a first interface, a second interface and a third interface; The first interface is used to connect to the outlet of the liquid phase separation device; the second interface is used to connect to the capillary glass spray needle to receive the analyte derived from the outlet of the liquid phase separation device, and to deliver the analyte to the gas phase separation device in the form of spray through the capillary glass spray needle; the third interface is used to connect to the non-denaturing solution, and when in use, the third interface is connected to a negative voltage to form a spray voltage loop; Optionally, the inner diameter of the outlet end of the capillary glass spray needle is in micrometer order; Optionally, the inner diameter of the capillary glass needle body is 150 μm-1 mm; Optionally, the third interface is arranged below the interface between the second joint and the capillary glass spray needle; Optionally, the interface device body further comprises a fourth interface for connecting the auxiliary modification liquid. When in use, the third interface is not connected to a negative voltage, and the fourth interface is connected to a negative voltage to form a spray voltage circuit; Optionally, the fourth interface is arranged below the side of the interface between the second joint and the capillary glass spray needle; Optionally, the interface device further comprises an interface support frame for supporting and mounting the interface device body; Optionally, the interface support frame is provided with an ion source identification contact for activating the mass spectrometer; Optionally, the non-denaturing solution is a non-denaturing sheath fluid; Optionally, the non-denaturing solution is a static sheath solution or a continuous flow sheath solution.

8. A combined instrument, characterized in that: It includes a liquid phase separation device and a gas phase separation device; The liquid phase separation device is connected to an interface device equipped with a capillary glass spray needle, the injection end of the liquid phase separation device is connected to a positive voltage to provide an electric field required to drive the solution to move, and the negative voltage is provided by the gas phase separation device, and the negative voltage is connected to the solution in the interface device, so that the injection end of the liquid phase separation device, the solution in the interface device and the electrospray form a stable electrical circuit; The interface device is connected to a non-denaturing solution.

9. The combined instrument according to claim 8, characterized in that: The liquid phase separation device is a capillary electrophoresis instrument or an instrument with similar functions; Optionally, the gas phase separation device is an ion mobility meter, a mass spectrometer containing an ion mobility module, a differential mobility analyzer, or an instrument with similar functions; Optionally, the interface device body comprises at least a first interface, a second interface and a third interface; The capillary outlet of the capillary electrophoresis instrument is connected to the first interface of the interface device, and the second interface of the interface device is connected to the capillary glass spray needle to receive the analyte guided out of the capillary outlet of the capillary electrophoresis instrument and send the analyte into the gas phase separation device in the form of spray through the capillary glass spray needle; The capillary injection end of the capillary electrophoresis instrument is connected to a positive voltage to provide an electric field required to drive the solution to move, and the negative voltage is provided by the gas phase separation device. The third interface is connected to a negative voltage, so that the injection end of the capillary electrophoresis instrument, the capillary, the solution in the interface device and the electrospray form a stable electrical circuit; Optionally, the inner diameter of the outlet end of the capillary glass spray needle is in micrometer order; Optionally, the inner diameter of the capillary glass needle body is 150 μm-1 mm; Optionally, the third interface is arranged below the interface between the second joint and the capillary glass spray needle; Optionally, the interface device body further comprises a fourth interface for connecting the auxiliary modification liquid. When in use, the third interface is not connected to a negative voltage, and the fourth interface is connected to a negative voltage to form a spray voltage circuit; Optionally, the fourth interface is arranged below the side of the interface between the second joint and the capillary glass spray needle; Optionally, the interface device further comprises an interface support frame for supporting and mounting the interface device body; Optionally, the interface support frame is provided with an ion source identification contact for activating the mass spectrometer; Optionally, the non-denaturing solution is a non-denaturing sheath fluid; Optionally, the non-denaturing solution is a static sheath solution or a continuous flow sheath solution.

10. Use of the combined instrument according to claim 8 or 9 in 1) synchronous separation of liquid phase conformation and gas phase conformation, 2) synchronous analysis of liquid phase conformation and gas phase conformation, or 3) synchronous analysis of the correlation between liquid phase conformation and gas phase conformation.

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