Interface device, combination instrument and application thereof

Abstract

The invention discloses an interface device, a combination instrument and application thereof. The interface device comprises an interface device body. The interface device body at least comprises a first interface, a second interface and a third interface. The first interface is used for communicating with an outlet end of a liquid phase separation device; the second interface is used for communicating with a capillary glass spray needle so as to receive the analyte guided out from the outlet end of the liquid phase separation device, and the analyte is fed into the gas phase separation device in a spray form through the capillary glass spray needle; the third interface is used for connecting a non-denatured solution, and is connected with negative voltage to form a spray voltage loop during use. When the interface device is used for combining a liquid-phase separation device and a gas-phase separation device, spray with good stability can be formed, so that the combined device can perform separation, analysis or correlation analysis on gas-phase conformation and liquid-phase conformation of an analyte in a non-denaturation state under a non-denaturation condition.

Description

Technical Field

[0001] The present application relates to the technical field of the combination of a liquid phase separation device and a gas phase separation device, and in particular to an interface device, a combination instrument and applications thereof. 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, it is difficult to collect fractions for offline IM analysis, so CE-IM needs to be combined online.

[0005] The CE-IM interface needs to be designed specifically for the low flow rate of CE itself, the requirements of IM for ionization efficiency and stability, and the need for maintaining the non-denaturing conditions of the protein solution in the combined application. Electrospray ionization (ESI) is currently the only ionization technology suitable for maintaining the original conformation of proteins. Existing interface design modes mainly include: (1) sheathless interface using a porous capillary tip as an ESI emitter; (2) sheath liquid CE-MS interface, in which the sheath liquid and nebulizing gas are coaxially injected into the CE effluent; (3) electroosmotic flow driven sheath flow interface; (4) auxiliary liquid interface introduced through a microinjection bottle.

[0006] All interfaces based on electrospray ionization technology must complete the spray voltage circuit. The existing ESI voltage application methods are: first, the voltage is directly contacted to the tip of the metal needle through a wire. This method has a slightly thick needle tip, resulting in low ionization efficiency, and often requires a denatured mobile sheath liquid to assist ionization. In addition, the metal needle is easily oxidized and difficult to clean and preserve. Second, a conductive layer is plated on the tip of the glass needle, but this method is too cumbersome and lacks stability. Third, an inert metal (such as platinum) electrode is inserted into the glass needle to apply the spray voltage. The disadvantage is that the reused electrode is prone to contamination, and electrochemical reactions are prone to occur at the interface between the electrode and the solution. Fourth, the above-mentioned type (4) interface uses an auxiliary modification liquid to send the sample into the mass spectrometer for detection. However, due to the particularity of the commercial ion mobility instrument, the vacuum degree in the ion source is relatively high, and the solution in the capillary glass needle is consumed rapidly, making it difficult to obtain a stable voltage circuit, and thus it is impossible to obtain a stable electrospray.

[0007] In addition, the electrospray stability of the above-mentioned (2) sheathless interface is not as good as that of the sheath flow interface. Therefore, in order to form a stable electrical contact between CE separation and ESI spray, a sheath liquid needs to be connected at the third joint. However, the flowing sheath liquid will greatly dilute the analyte, thereby reducing the detection sensitivity. The above-mentioned (3) interface has better compatibility in CE-MS coupling applications. In addition, the denaturing sheath liquid is generally used for auxiliary ionization, but it also destroys the non-denaturing environment of the analyte.

[0008] In summary, existing detection schemes and interfaces are difficult to meet the requirements of correlation analysis between protein solution phase conformation and gas phase conformation. Summary of the invention

[0009] The purpose of the present application is to provide an improved interface device, a coupling instrument and applications thereof.

[0010] In order to achieve the above objectives, this application adopts the following technical solutions:

[0011] One aspect of the present application discloses an interface device, including an interface device body, the interface device body including at least a first interface, a second interface and a third interface; the first interface is used to connect to the outlet end of a liquid phase separation device; the second interface is used to connect to a capillary glass spray needle to receive the analyte derived from the outlet end of the liquid phase separation device, and to deliver 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 to a non-denaturing solution, and when in use, the third interface is connected to a negative voltage to form a spray voltage circuit.

[0012] It should be noted that the interface device of the present application can be adapted to a commercial gas phase separation device, which is connected to a liquid phase separation device, so as to realize the combination of the liquid phase separation device and the gas phase separation device under non-denaturing conditions. Among them, commercial gas phase separation devices include but are not limited to ion mobility instruments and mass spectrometers containing ion mobility modules, and liquid phase separation devices include but are not limited to capillary electrophoresis instruments. The interface device of the present application has good spray stability, so that the combined device of liquid phase separation and gas phase separation can separate, analyze or correlate the gas phase conformation and liquid phase conformation of the non-denaturing state of the analyte under non-denaturing conditions.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] In one implementation of the present application, the interface device further includes an interface support frame for supporting and installing the interface device body.

[0019] In one implementation of the present application, an ion source identification contact is provided on the interface support frame for activating the mass spectrometer.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] In one implementation of the present application, the non-denaturing solution is a non-denaturing sheath fluid.

[0026] In one implementation of the present application, the non-denaturing solution is a static sheath solution or a continuous flow sheath solution.

[0027] In one implementation of the present application, the interface device is used to perform liquid phase conformation and gas phase conformation separation on the analyte.

[0028] In one implementation of the present application, the interface device is used to perform liquid phase conformation analysis and gas phase conformation analysis on the analyte.

[0029] In one implementation of the present application, the interface device is used to perform correlation analysis between the liquid phase conformation and the gas phase conformation of the analyte.

[0030] It should be noted that, in the present application, the interface device is used to perform liquid phase conformation and gas phase conformation separation, analysis or correlation analysis on the analyte, which means that the liquid phase separation device and the gas phase separation device are combined by using the interface device.

[0031] Another aspect of the present application discloses a combined instrument, which includes the interface device of the present application.

[0032] It should be noted that the combined instrument of the present application, due to the use of the interface device of the present application, can form a stable spray during use, and can separate, analyze or perform correlation analysis on the gas phase conformation and liquid phase conformation of the non-denaturing state of the analyte under non-denaturing conditions.

[0033] In one implementation of the present application, the combined instrument of the present application is a combined instrument of a liquid phase separation device and a gas phase separation device.

[0034] In one implementation of the present application, the liquid phase separation device is a capillary electrophoresis instrument or an instrument with similar functions.

[0035] 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.

[0036] In one implementation of the present application, the capillary outlet end 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 exported from the capillary outlet end 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, 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.

[0037] 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.

[0038] In one implementation of the present application, the inner diameter of the capillary glass spray needle body is 150 μm-1 mm.

[0039] 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.

[0040] 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.

[0041] Another aspect of the present application discloses an assembly method of the combined instrument of the present application, including inserting the inlet end of the liquid phase separation device into a sample bottle, and placing the outlet end through a first interface and into the needle tip of a capillary glass spray needle connected to a second interface; installing the interface device on a gas phase separation device, and replacing the original spray needle of the gas phase separation device with the capillary glass spray needle connected to the second interface.

[0042] Among them, a capillary glass spray needle connected to the second interface is used to replace the original spray needle of the gas phase separation device. For example, the baffle, conductive wire and spray needle device of the gas phase separation device, such as a mass spectrometer containing an ion mobility module, are removed, the interface device is installed at the corresponding position of the mass spectrometer, and the capillary glass spray needle of the second connector is used to replace the original spray needle.

[0043] Another aspect of the present application discloses a method for separating liquid phase conformation and gas phase conformation, including using the combined instrument of the present application to separate liquid phase conformation and gas phase conformation.

[0044] Another aspect of the present application discloses a method for liquid phase conformation and gas phase conformation analysis, including using the combined instrument of the present application to perform liquid phase conformation and gas phase conformation analysis.

[0045] Another aspect of the present application discloses a method for correlation analysis of liquid phase conformation and gas phase conformation, including using the combined instrument of the present application to perform correlation analysis of liquid phase conformation and gas phase conformation.

[0046] Due to the adoption of the above technical solution, the beneficial effects of this application are:

[0047] The interface device of the present application, when used in conjunction with a liquid phase separation device and a gas phase separation device, can form a spray with good stability, so that the combined device can separate, analyze or perform correlation analysis on the gas phase conformation and liquid phase conformation of the analyte in a non-denaturing state under non-denaturing conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a top cross-sectional view of the interface device body in the embodiment of the present application;

[0049] Figure 2 is a positional relationship diagram of the interface device body and the interface support frame in the embodiment of the present application;

[0050] Figure 3 This is a schematic diagram of the CE-IM-MS technology in the embodiments of the present application;

[0051] Figure 4 It is the MT map and protein spectra of the Pierce mixed protein sample in the examples of this application;

[0052] Figure 5 It is the MT map and CCS map of each protein in the Pierce mixed protein sample in the example of this application;

[0053] Figure 6 It is a DT diagram corresponding to the specific charge state and specific MT of each protein in the Pierce mixed protein sample in the embodiment of the present application. DETAILED DESCRIPTION

[0054] In response to the existing interface design mode, 1) the metal spray needle ionization efficiency is low, it is easy to oxidize, and it is difficult to clean and preserve; 2) the conductive layer plated on the tip of the glass spray needle is not stable enough; 3) the electrospray stability is poor; 4) a large amount of flowing sheath liquid dilutes the analyte and reduces the detection sensitivity. This application creatively develops a new interface device.

[0055] The interface device of the present application 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.

[0056] It should be noted that the novel interface device of the present application is suitable for CE-IM (including CE-IM-MS). In one implementation of the present application, a fine-caliber tip glass capillary is used as an electrospray ionization needle to efficiently ionize intact proteins at low flow rates, and a non-denaturing sheath fluid is used to maintain the original conformation of the protein. The non-denaturing sheath fluid used in the interface device of the present application not only realizes the formation of stable electrical contact between CE separation and ESI spray and improves the detection sensitivity, but also retains the non-denaturing conformation of the analyte. Among them, the sheath fluid forms include static sheath fluid and continuous flow sheath fluid,

[0057] Static sheath liquid avoids problems such as spray instability caused by flowing sheath liquid and decreased sensitivity due to dilution. 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 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, low-flow continuous flow sheath liquid has the advantages of strong flow rate controllability and more stable spray. In addition, low-flow continuous flow sheath liquid can avoid changes in solution parameters because it is supplemented with fresh solution. Solution parameters include but are not limited to pH, ionic strength, salt concentration, concentration of other components, etc.; it overcomes 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 static sheath liquid, and effectively prevents artificial changes in protein conformation. Therefore, static sheath liquid or low-flow 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.

[0058] Furthermore, considering the particularity of the ion mobility mass spectrometer, the sheath liquid and / or auxiliary modification liquid are designed to be located below the interface, avoiding the problem of rapid consumption of the solution in the device due to high vacuum. The present application combines CE, IM, and MS methods to obtain the mass-to-charge ratio (m / z), signal intensity (intensity), gas phase drift time (drift time, DT), liquid phase migration time (migration time, MT), and four-dimensional data information of the sample, and realizes the correlation analysis of the liquid phase conformation and gas phase conformation of the protein.

[0059] 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.

[0060] Example

[0061] In order to realize the coupling analysis of CE with IM or IM-MS, including the analysis of intact proteins under non-denaturing conditions, a new interface device was developed in this study.

[0062] 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 solution phase; MS can be used for the determination of the molecular weight and ion valence of proteins.

[0063] The interface device of 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 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, which is used to provide a stable continuous liquid flow as the sheath liquid of the CE effluent. On the one hand, the increase in the total flow rate after merging with the CE effluent improves the electrospray stability, and the low flow rate also avoids the sample dilution problem. On the other hand, it overcomes the pH change problem caused by the accumulation of a single charged electrolyte in the static sheath liquid pool during the continuous operation of CE in the existing design, and effectively prevents the artificial change of protein conformation. Therefore, static sheath liquid or continuous flow sheath liquid can be selectively used according to experimental requirements. In this example, static sheath liquid is used; the ionization module uses a glass capillary with a fine-caliber tip as a spray needle to ensure efficient ionization of intact proteins at a low flow rate.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] The specific instruments and materials used in this case include:

[0069] Ion mobility quadrupole time-of-flight mass spectrometer (SYNAPT XS Ion Mobility Time-of-Flight Mass Spectrometer, Waters); capillary electrophoresis separation instrument (CESI 8000 Plus High Performance Separation-ESI Module, AB Sciex).

[0070] 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.

[0071] The principle of the interface device in this example:

[0072] The sample and background electrolyte (BGE) solution enter the CE capillary under a certain pressure. Figure 3As shown in the figure, in capillary electrophoresis, the charged substance itself will move relative to the background electrolyte solution due to the electrophoretic effect. The direction of the capillary electrophoresis velocity depends on the positive or negative charge of the analyte in the background electrolyte solution. When the isoelectric point (pI) value 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. 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 the traveling wave ion mobility spectrometry (TWIMS) mass spectrometry, the ions form a motion trajectory similar to a traveling wave due to the horizontal DC voltage and the vertical periodic radio frequency voltage applied in the mobility cell. Due to the presence of inert gas in the mobility cell, ions with a more compact structure are subject to less resistance and have a shorter movement time, thereby achieving the separation of ions with different structures. CE-IM-MS coupling technology combines the above two separation technologies to achieve the separation and detection of gas phase and liquid phase structures at the same time.

[0073] Testing process:

[0074] When the background electrolyte solution enters the capillary glass needle from the CE capillary, the modification solution will form an electrospray voltage loop with the analyte 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 can separate ions of different charges, sizes and shapes in the mobility cell through inert gas collision and electric field induction, and the mass spectrometer can determine its precise molecular weight, and finally realize the separation and detection of analytes in liquid and gas phase environments.

[0075] Application example: Interface feasibility testing

[0076] Steps:

[0077] Install the interface device body: The interface device body includes a four-way joint, which includes a first joint, a second joint, a third joint and a fourth joint. Figure 1As shown, the first connector 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 connector with the PEEK threaded adapter 4; the capillary glass spray needle 2 is installed on the second connector, and similarly, the capillary glass spray needle 2 is fixed with the FEP sleeve 3 and then installed to the second connector with the PEEK threaded adapter; the third connector is connected to the non-denatured static sheath liquid, and the fourth connector 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 non-denatured 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.

[0078] 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.

[0079] 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 device body on the interface support plate; wherein, the ion source identification contacts are a sensor for identifying the ion source that comes with Synapt XS. Device inspection: use a 200μL syringe to inject the background electrolyte into the interface device along the static sheath liquid end. If there is a 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 wire, and 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.

[0080] Experimental conditions:

[0081] (1) Reagents

[0082] Pierce protein mixture was purchased from Thermo Fisher (USA). It is composed of 6 simple proteins with molecular weights ranging from 9kDa to 70kDa, including human IGF-1LR3 (IGF) (P05019, 40-118), human thioredoxin (TRX) (Q99757, 60-166), Streptococcus dysgalactiae protein G (PG) (P06654, 223-413), bovine carbonic anhydrase 1 (CA) (P00921), Streptococcus protein AG (PAG) (P02976, P19909) and Escherichia coli Exo Klenow (P00582, 324-928); hydroxypropyl cellulose (HPC), ammonium acetate (NH 4 Ac, HPLC grade) were of chromatographic grade purity and purchased from Sigma-Aldrich (Sigma-Aldrich, USA).

[0083] (2) Sample processing

[0084] Weigh the Pierce protein and dissolve it in ultrapure water. Select a 3kDa ultrafiltration membrane and replace the solution with 20mM ammonium acetate solution to remove the non-volatile salts in the protein that are incompatible with mass spectrometry. 4 The Ac solution was centrifuged at 12000 g for 5 min to activate the filter membrane; then the sample and 20 mM NH 4 The Ac solution was centrifuged at 12000g for 10 min; the filtrate was collected after repeated centrifugation 5-6 times. Finally, the protein concentration after desalting was measured.

[0085] (3) CE-IM-MS experimental conditions and parameter settings

[0086] The capillary used was a 100 cm long HPC coated tube, and the BGE was 20 mM NH 4 Ac solution, injection volume was 2psi10s, separation voltage was 30kV, and the pressure was maintained at 0.5psi during the separation process.

[0087] The Pierce protein was placed in a CE sample bottle and entered into the mass spectrometer for detection after capillary separation. The main parameter conditions of the Synapt XS mass spectrometer were set as follows: Capillary voltage 1.2-1.7 kV, Source Temperature 120 °C, Trap Collision Energy 10 V.

[0088] Experimental data:

[0089] Data analysis method: MassLynx V4.2 software (Waters, USA), DriftScope V2.9 (Waters, USA), CIUSuite 2 (GitHub, USA).

[0090] The test results are as follows Figures 4 to 6 As shown, Figure 4 Figure A is the total ion current diagram of the Pierce protein sample, showing the protein CE migration time (MT). Figures B to G are mass spectra, showing the molecular weight, valence state and MT corresponding to the mass spectrum peak of the specific valence state of each protein in the sample. Figure 5 Figure A is the extracted ion current diagram. The MT diagram measured by CE separation shows the liquid phase migration time of each protein in the sample. Figure B is the CCS diagram. The CCS diagram measured by IM separation reflects the gas phase collision cross-sectional area of ​​each protein in the sample. Figure 6 It is the DT diagram under the specific valence state mass spectrum peak and specific MT of each Pierce protein, showing the four-dimensional data information of the CE-IM-MS coupling method.

[0091] Figures 4 to 6 The results show that the CE-IM-MS connected by the interface device of this example can perform liquid phase conformation and gas phase conformation correlation analysis on Pierce protein, and obtain four-dimensional data information of protein sample including mass-to-charge ratio (m / z), signal intensity (intensity), gas phase drift time (drift time, DT) and liquid phase migration time (migration time, MT).

[0092] In summary, the interface device of this example (1) is easy and convenient to install by CE-IM-MS coupling; (2) CE-IM-MS coupling technology can separate and detect protein mixed samples with high stability and resolution; (3) this interface device realizes the gas phase and liquid phase separation of each protein in the Pierce protein sample in one detection; (4) this interface device obtains the four-dimensional data information of m / z, intensity, DT, and MT of each protein.

[0093] 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. An interface device, characterized in that: The interface device body 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 derived from the outlet end of the liquid phase separation device, and to deliver 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 to a non-denaturing solution. When in use, the third interface is connected to a negative voltage to form a spray voltage circuit.

2. The interface device according to claim 1, characterized in that: The third interface is arranged at the lower side of 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 at a side and below the interface between the second joint and the capillary glass spray needle.

3. The interface device according to claim 1, characterized in that: The interface device also includes an interface support frame for supporting and installing the interface device body; Optionally, an ion source identification contact is provided on the interface support frame for activating the mass spectrometer.

4. The interface device according to any one of claims 1 to 3, characterized in that: The analytes include natural molecules or synthetic molecules with the same molecular formula but 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 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; Optionally, the interface device is used to perform liquid phase conformational and gas phase conformational separation on the analyte; Optionally, the interface device is used to perform liquid phase conformation analysis and gas phase conformation analysis on the analyte; Optionally, the interface device is used to perform liquid phase conformation and gas phase conformation correlation analysis on the analyte.

5. A combined instrument, characterized in that: The invention comprises the interface device according to any one of claims 1 to 4.

6. The combined instrument according to claim 5, characterized in that: A combined instrument for 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 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 deliver the analyte to 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, 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; 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.

7. The method for assembling the combined instrument according to claim 5 or 6, characterized in that: The method comprises inserting the inlet end of the liquid phase separation device into a sample bottle, and placing the outlet end through a first interface into the needle tip of a capillary glass spray needle connected to a second interface; The interface device is installed on the gas phase separation device, and the capillary glass spray needle connected to the second interface is used to replace the original spray needle of the gas phase separation device.

8. A method for separating liquid phase conformation and gas phase conformation, characterized in that: The method comprises using the combined instrument described in any one of claims 5 to 7 to separate liquid phase conformation and gas phase conformation.

9. A method for analyzing liquid phase conformation and gas phase conformation, characterized in that: The method comprises using the combined instrument described in any one of claims 5 to 7 to perform liquid phase conformation analysis and gas phase conformation analysis.

10. A method for analyzing the correlation between liquid phase conformation and gas phase conformation, characterized in that: The method comprises using the combined instrument described in any one of claims 5 to 7 to perform correlation analysis of liquid phase conformation and gas phase conformation.

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