Mass spectrum sampling integrated device for space-time synchronous analysis of complete protein and enzymolysis polypeptide and analysis method

Abstract

The invention relates to a mass spectrum sampling integrated device for space-time synchronous analysis of intact protein and enzymolysis polypeptide and an analysis method, and belongs to the technical field of mass spectrum analysis. The device aims to solve the problem that synchronous analysis of complete protein and enzymolysis polypeptide is difficult to realize in the prior art. The device comprises a sample inlet, a first six-way valve, a second six-way valve, a four-way valve, a pancreatin column, a complete protein separation column C4 and a polypeptide separation column C18a. A first passage consisting of a sample inlet, a first six-way valve and a quantitative loop is formed; the second passage consists of a sample inlet, a second six-way valve, a pancreatin column, a capture column C18, a four-way valve and a polypeptide separation column C18a; and the third passage consists of a mobile phase inlet, a first six-way valve, a quantitative loop, a four-way valve and a complete protein separation column C4. The method is mainly used for realizing time-space synchronous analysis of complete protein and enzymolysis polypeptide in mass spectrometry.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry analysis. More specifically, the present invention relates to a mass spectrometry injection integration device and an analysis method for the spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides. Background Art

[0002] In the technical field of mass spectrometry analysis, the analysis of intact proteins and enzymatically digested polypeptides is of crucial significance for in-depth understanding of the structure, function, and related biological processes of proteins. However, for a long time, achieving the spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides has been a major challenge in this field.

[0003] Traditional mass spectrometry analysis methods often perform the analysis processes of intact proteins and enzymatically digested polypeptides separately. This is because there are significant differences in the properties of intact proteins and enzymatically digested polypeptides. Intact proteins have large molecular weights and complex structures, while enzymatically digested polypeptides have relatively small molecular weights and relatively simple structures. They require different conditions and technical means in separation, detection, and other steps. Separated operation not only increases the complexity and time cost of the experiment, but also, due to the independence of the two analysis processes, it is difficult to ensure the consistency in time and space, resulting in the inability to accurately obtain information on both at the same moment and in the same system, thus affecting the accurate interpretation of the overall characteristics of proteins.

[0004] From the perspective of technical principles, the separation of intact proteins usually requires a separation column that can adapt to its large molecular weight and complex structure, such as a C4 separation column, to achieve effective separation. Enzymatically digested polypeptides are more suitable for separation using a C18 separation column. However, in traditional injection and analysis systems, it is difficult to reasonably integrate separation columns and related processes for substances with different properties, making it a difficult problem to efficiently process intact proteins and enzymatically digested polypeptides simultaneously in the same device.

[0005] In addition, when processing samples, the existing technology lacks effective means to orderly and synchronously guide and process intact proteins and enzymatically digested polypeptides in the sample. For example, it is difficult to precisely control the injection and separation timing of intact proteins while ensuring the smooth progress of the enzymatic digestion process, making the analysis of the two unable to be carried out synchronously. Moreover, during the injection process, due to the lack of a reasonable pathway design, it is impossible to precisely control different mobile phases and sample flows, resulting in the difficulty for intact proteins and enzymatically digested polypeptides to reach an ideal synchronous state when entering the separation column and the mass spectrometer, thereby affecting the accuracy and reliability of the analysis results.

[0006] In summary, there are many deficiencies in the existing technology in the spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides, and there is an urgent need for a new technical solution to solve these problems to improve the efficiency and accuracy of mass spectrometry analysis in the field of protein research. Summary of the Invention

[0007] One object of the present invention is to provide a mass spectrometry injection integrated device and method for synchronous analysis of intact proteins and enzymatically digested polypeptides in space and time, which can integrate the analysis processes of intact proteins and enzymatically digested polypeptides in the same device and achieve synchronous analysis of the two within the same time frame.

[0008] The mass spectrometry injection integrated device for synchronous analysis of intact proteins and enzymatically digested polypeptides in space and time according to the present invention includes: a sample inlet;

[0009] A first six-way valve, the sample inlet is connected to the first six-way valve, and a quantitative loop is provided in the first six-way valve;

[0010] A second six-way valve, which is connected to the first six-way valve, a mobile phase inlet is connected to the second six-way valve, and a capture column C18 is provided in the second six-way valve;

[0011] A four-way valve, which is connected to both the first six-way valve and the second six-way valve, and the four-way valve is also connected to an intact protein separation column C4 and a polypeptide separation column C18a; and

[0012] A trypsin protein column, which is provided between the first six-way valve and the second six-way valve;

[0013] Among them, the following channels are formed:

[0014] The first channel consists of the sample inlet, the first six-way valve, and the quantitative loop;

[0015] The second channel consists of the sample inlet, the second six-way valve, the trypsin protein column, the capture column C18, the four-way valve, and the polypeptide separation column C18a; and

[0016] The third channel consists of the mobile phase inlet, the first six-way valve, the quantitative loop of the first six-way valve, the four-way valve, and the intact protein separation column C4.

[0017] Preferably, the mass spectrometry injection integrated device for synchronous analysis of intact proteins and enzymatically digested polypeptides in space and time according to the present invention further includes:

[0018] A first liquid phase pump, which is connected to the sample inlet; and

[0019] A second liquid phase pump, which is connected to the mobile phase inlet;

[0020] Among them, the first six-way valve, the second six-way valve, and the four-way valve are connected through capillary pipelines.

[0021] Preferably, the method for synchronous analysis of intact proteins and enzymatically digested polypeptides in space and time according to the present invention includes the following steps:

[0022] Inject a protein sample into a capillary quartz tube and dock it to the sample inlet;

[0023] Adjust the first six-way valve to form the first passage; inject a part of the sample in the capillary quartz tube into the sample inlet at a flow rate of 0.4 - 0.6 uL / min until it reaches the quantitative loop of the first six-way valve;

[0024] After that, adjust the first six-way valve again and adjust the second six-way valve to form the second passage, and inject the remaining sample into the trypsin column through the sample inlet;

[0025] After that, adjust the first six-way valve again and adjust the four-way valve to form the third passage, inject a part of the sample in the quantitative loop into the intact protein separation column C4 connected to the four-way valve through the mobile phase inlet at a flow rate of 2 - 4 uL / min, and rinse for 8 - 12 min for desalting; meanwhile, the remaining sample is subjected to on-line enzymatic digestion on the trypsin column;

[0026] After that, adjust the first six-way valve again, adjust the second six-way valve, and adjust the four-way valve to form the second passage, and inject the enzymatically digested polypeptide sample into the polypeptide capture column C18;

[0027] Finally, introduce the processed samples in the intact protein separation column C4 and the polypeptide capture column C18 into the mass spectrometer for detection to obtain the intact protein and enzymatically digested polypeptide data information of the same sample.

[0028] Preferably, for the method for synchronous spatio-temporal analysis of intact protein and enzymatically digested polypeptide of the present invention, the protein sample is the protein of Escherichia coli BL21 strain, and its preparation method is:

[0029] Place the Escherichia coli BL21 strain in 120 - 160 mL of LB medium, and culture it in a constant temperature shaker at 36 - 37 °C for 11 - 13 h until the final OD600 value of the bacteria is 2.1 - 2.3;

[0030] Transfer the cultured bacterial liquid to a 50 - 60 mL centrifuge tube, and centrifuge it at a speed of 4000 - 6000 rpm for 8 - 12 min to obtain a precipitate;

[0031] Resuspend the above precipitate in 5 - 6 mL of binding buffer, and ultrasonically disrupt the resuspended bacteria under ice bath conditions;

[0032] After the bacteria are disrupted, centrifuge it at a speed of 14000 - 15000 rpm for 15 - 25 min to obtain the supernatant;

[0033] Filter the obtained supernatant through a 0.22 um aqueous filter membrane;

[0034] Use the BCA method to measure the protein concentration of the supernatant after filtration through the filter membrane, and adjust the final protein concentration to 0.7 - 0.9 mg / mL using the binding buffer;

[0035] Before loading the sample, add urea to the protein sample to make the final concentration of urea 8 - 9 M, and then place the sample in a 37 - 38 °C metal bath and incubate for 5 - 6 min; and

[0036] After incubation, immediately dilute the sample with 25 mM ammonium bicarbonate solution to a urea concentration of 1 M to obtain the sample.

[0037] Preferably, in the method for synchronous spatio - temporal analysis of intact protein and enzymatically digested polypeptide of the present invention, the enzymatic digestion process includes: changing the mobile phase of the first liquid phase pump connected to the sample inlet to water, setting the flow rate to 1 - 1.5 μL / min, flushing the trypsin column for 50 - 70 min, so that the polypeptide products enzymatically digested by the trypsin column are loaded onto the capture column C18, and waiting for the end of the intact protein analysis process.

[0038] Preferably, in the method for synchronous spatio - temporal analysis of intact protein and enzymatically digested polypeptide of the present invention, set the initial mobile phase ratio for polypeptide analysis of the second liquid phase pump connected to the mobile phase inlet, so that the ratio of water to acetonitrile is 98%:2%;

[0039] After that, perform polypeptide separation according to a specific mobile phase gradient. Depending on the complexity of the sample, within 25 - 100 min, gradually increase the proportion of acetonitrile from 2% to 35%, and the polypeptides are gradually separated and eluted into the mass spectrometry detection. Depending on the complexity of the sample and the separation requirements, the flow rate is set to 0.5 - 1.5 μL / min.

[0040] Preferably, in the method for synchronous spatio - temporal analysis of intact protein and enzymatically digested polypeptide of the present invention, set the initial mobile phase ratio for polypeptide analysis of the second liquid phase pump connected to the mobile phase inlet, so that the ratio of water to acetonitrile is 98%:2%;

[0041] After that, perform complete separation according to a specific mobile phase gradient. Depending on the complexity of the sample, within 25 - 80 min, gradually increase the proportion of acetonitrile from 2% to 40%, and the intact proteins are gradually separated and eluted into the mass spectrometry detection; depending on the complexity of the sample and the separation requirements, the flow rate is set to 1.0 - 1.5 μL / min.

[0042] Preferably, in the method for synchronous spatio - temporal analysis of intact protein and enzymatically digested polypeptide of the present invention, the protein sample is denatured, including the following steps:

[0043] Prepare 1 mg / mL cytochrome C, 1 mg / mL hemoglobin and 2 mg / mL bovine serum albumin respectively, and the solvent is phosphate buffer;

[0044] Take 50 μL of each of the above three protein solutions respectively, add 50 μL of 8 M urea and mix to denature the proteins to obtain the final protein sample.

[0045] Preferably, for the method for synchronous spatio-temporal analysis of intact protein and enzymatically hydrolyzed polypeptide of the present invention, the enzymatic digestion step includes:

[0046] Add 900 uL of 25 mM ammonium bicarbonate solution to the denatured protein solution for dilution so that the urea concentration in the protein solution is lower than 1 M;

[0047] Use a quartz capillary with a length of 32 cm and an inner diameter of 200 um to siphon the protein solution. Connect both ends of the capillary filled with the protein solution to the outlet of the first liquid phase pump, the trypsin immobilization column, and the capture column C18 respectively;

[0048] Under the push of the first liquid phase pump, the protein sample passes through the trypsin column and the capture column C18 in sequence, so that the protein sample is captured in the capture column C18 after being enzymatically digested into polypeptides;

[0049] Among them, the flow rate of the first liquid phase pump is set to 0.5 uL / min, and the injection time is 12 - 16 min.

[0050] Preferably, the method for packing the trypsin column of the present invention includes:

[0051] Take 200 uL of potassium silicate solution and mix it with 20 uL of formamide, and vortex mix at a speed of 3000 rpm;

[0052] Intercept a quartz capillary with an inner diameter of 200 um, an outer diameter of 370 um, and a length of 15 cm, insert it into the mixed solution of potassium silicate and formamide, suck the solution to a height of 1 cm inside the capillary, and then put the capillary into an oven at 100 °C and heat it for 12 h to manufacture a column packing sieve plate;

[0053] Dissolve the immobilized trypsin packing in 100 mM acetic acid solution to prepare a solution with a concentration of about 1 mg / mL; use a capillary column packing instrument to fill the above chromatographic packing solution into the sieve plate capillary obtained in step one, and the filling length is 5 cm;

[0054] Use a micro-injection pump to inject ammonium bicarbonate solution with pH = 8 into the immobilized trypsin column at 4 °C, and set the flow rate to 2 uL / min to wash away the excess acetic acid in the column and balance the trypsin column.

[0055] Beneficial effects

[0056] Through a unique multi-channel design, the device can ingeniously integrate the analysis processes of intact protein and enzymatically hydrolyzed polypeptide in the same device. It realizes the synchronous analysis of the two within the same time frame, greatly saving the analysis time and sample consumption. It avoids the problems of cumbersome operation and easy introduction of errors in the traditional method that requires separate analysis of intact protein and enzymatically hydrolyzed polypeptide, and improves the analysis efficiency and accuracy.

[0057] A first liquid-phase pump and a second liquid-phase pump are added to accurately control the delivery of the sample and the mobile phase respectively. This ensures that the flow rate and flow volume of the sample and the mobile phase in the device are stable and accurate, making the entire analysis process more reliable. The use of capillary tubing connections reduces the dead volume of the tubing, further optimizing the transmission of the sample and the mobile phase between valves and improving the overall performance of the device.

[0058] This method utilizes the device in claim 1 in detail and orderly. By precisely controlling parameters such as the switching of each passage and the sample flow rate, the spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides is achieved. From sample injection to final mass spectrometry data analysis, each step is closely connected, ensuring the coherence and efficiency of the analysis process and effectively improving the reliability and repeatability of the analysis results.

[0059] A preparation method specifically for proteins of Escherichia coli BL21 strain, with each step optimized from strain culture to final sample acquisition. It can ensure the stable quality of the prepared protein sample, and its characteristics meet the requirements of subsequent spatio-temporal synchronous analysis. The standardized preparation process reduces the analysis errors caused by sample differences, providing a reliable sample basis for subsequent accurate analysis.

[0060] Specific mobile phase replacement and rinsing conditions optimize the enzymatic digestion process. While ensuring the enzymatic digestion effect, the remaining sample is accurately loaded onto the capture column C18 and coordinated with the intact protein analysis process. This avoids problems such as enzyme inactivation, incomplete enzymatic digestion, or over-enzymatic digestion that may occur during the enzymatic digestion process, improving the quality of the enzymatic digestion products and the accuracy of subsequent analysis.

[0061] A reasonable initial mobile phase ratio for polypeptide analysis and a specific mobile phase gradient setting enable efficient separation of polypeptides within 25 - 100 min and accurate entry into mass spectrometry detection. The optimized mobile phase conditions improve the resolution and separation effect of polypeptide separation, avoiding problems such as incomplete polypeptide separation or detection signal interference caused by improper mobile phase settings, providing a guarantee for accurate polypeptide mass spectrometry analysis.

[0062] Both the mobile phase conditions for polypeptide separation are optimized, and the gradient and flow rate for intact protein separation are set. This ensures that both intact proteins and polypeptides can be effectively separated, eluted, and accurately enter mass spectrometry detection under their respective appropriate conditions. The two analysis processes are coordinated with each other, improving the overall analysis efficiency and the accuracy of the results, and enabling the simultaneous acquisition of high-quality intact protein and polypeptide analysis data.

[0063] This denaturation method is specific to a particular protein sample. By a specific method of preparing the protein solution and mixing it with urea, the protein is effectively denatured. This creates favorable conditions for subsequent enzymatic digestion and analysis processes, improving the reaction activity and analysis effect of the protein in subsequent steps and ensuring the smooth progress of the analysis process.

[0064] Precisely controlled enzymatic digestion conditions, including the dilution solution, the time for the sample to flow through the trypsin column, etc., enable the protein sample to be efficiently digested into polypeptides and accurately captured in the C18 capture column. The operation is simple and highly stable, avoiding errors and losses during the enzymatic digestion process, improving the acquisition efficiency and quality of the enzymatic digestion products, and providing a reliable sample source for subsequent polypeptide analysis.

[0065] A new method for packing the trypsin column, with each step carefully designed from the manufacture of the column packing sieve plate to the filling of the packing and subsequent equilibration. It can improve the packing quality of the trypsin column and ensure stable column performance. A high-quality trypsin column is conducive to improving the enzymatic digestion efficiency and effect, thereby enhancing the accuracy and reliability of the entire spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides. Brief Description of the Drawings

[0066] Figure 1 Schematic diagram of the mass spectrometry injection integration device for the spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides in this application;

[0067] Figure 2 Test chart of the effect of trypsin column enzymatic digestion of protein samples according to an embodiment of the present invention;

[0068] Figure 3 Intact protein mass spectrometry signal of hemoglobin obtained by the integration device according to an embodiment of the present invention;

[0069] Figure 4 Chromatogram of enzymatically digested hemoglobin protein obtained by the integration device according to an embodiment of the present invention;

[0070] Figure 5 Analysis of BL21 bacterial lysate by the integration device according to an embodiment of the present invention. Detailed Description of the Invention

[0071] The following further elaborates on the present invention with reference to the accompanying drawings, so that those skilled in the art can implement it according to the description in the specification.

[0072] As Figures 1 - 5 shown, in Figure 1In (a), after the sample is introduced into the capillary, it enters the 7 μL quantitative loop under the push of the liquid phase pump 1. (b) After the quantitative loop is filled with the sample, the preset second liquid phase pump flushes the C4 capillary column with water as the mobile phase for 10 min for desalting. Subsequently, the proportion of acetonitrile in the mobile phase of liquid phase pump 2 gradually increases, and the mass spectrometry is turned on for data acquisition to achieve the analysis of intact proteins. At the same time, liquid phase pump 1 pushes the remaining sample to the trypsin column and the 5 cm long C18 column. The protein sample completes enzymatic digestion and the capture of polypeptides after enzymatic digestion. The mobile phase of liquid phase pump 1 is changed to a high proportion of aqueous phase to introduce the enzymatically digested polypeptides into the C18 capture column and elute the salts in the flow path, which flows out from the 4th outlet of the six-way valve on the left. (c) After the detection of intact proteins is completed synchronously with the enzymatic digestion, polypeptide capture, and desalting of another flow path, through the preset liquid phase method program, the six-way valve on the right is adjusted from position B to position A, the six-way valve on the left is adjusted from position A to position B, the four-way valve is adjusted to position 2, and the proportion of the organic phase in the mobile phase of the liquid phase pump gradually increases. The polypeptides in the 5 cm C18 column are gradually eluted and separated by the 15 cm C18 chromatographic column to achieve the detection of polypeptides after enzymatic digestion.

[0073] In Figure 2 it shows the test of the effect of enzymatic digestion of protein samples by the trypsin column. For cytochrome C, hemoglobin, and bovine serum albumin samples, 10, 17, and 31 polypeptides are detected respectively (marked as the detected polypeptide signal peaks), and the sequence coverages are 71.4%, 73.9%, and 48.4% respectively.

[0074] In Figure 3 it shows the intact protein mass spectrometry signal of hemoglobin obtained by the integrated analysis device; (a) is the chromatogram of the hemoglobin signal, and (b) and (c) are the mass spectrometry signal diagrams of the detected hemoglobin α subunit and β subunit respectively.

[0075] In Figure 4 it shows the chromatogram of the enzymatically digested hemoglobin obtained by the integrated device; (marked as the chromatographic peaks containing the signals of enzymatically digested hemoglobin polypeptides, and a total of 15 target polypeptides are identified)

[0076] In Figure 5 the integrated device analyzes the BL21 bacterial lysate. (a) The chromatogram of intact protein separation, (b) the chromatogram of enzymatically digested polypeptide analysis, (c) the molecular weight distribution diagram of intact proteins detected by the TD flow path, (d) the Venn diagram of proteins detected by TD and BU.

[0077] The present invention provides a mass spectrometry injection integration device and method for the spatial and temporal synchronous analysis of intact proteins and enzymatically digested polypeptides.

[0078] This embodiment relates to a mass spectrometry injection integration device for the synchronous spatio-temporal analysis of intact proteins and enzymatically digested polypeptides. The device mainly consists of a sample inlet, a first six-way valve 2, a second six-way valve 1, a four-way valve 3, a trypsin protein column 6, and other components.

[0079] The sample inlet, as the starting end of the entire device, is used to access the protein sample to be analyzed. The protein sample can be docked with it through a capillary quartz tube or other means. The first six-way valve 2 is connected to the sample inlet, and a quantitative loop 4 is provided inside it. The function of the quantitative loop 4 is to accurately measure a certain volume of the sample for subsequent accurate analysis. When the device is in a specific working state, the sample can enter from the sample inlet and flow into the quantitative loop 4 through the path selection of the first six-way valve 2.

[0080] The second six-way valve 1 is connected to the first six-way valve 2, and at the same time, the mobile phase inlet is connected to the second six-way valve 1. A capture column C18 5 is provided in the second six-way valve 1. The capture column C18 5 is mainly used to capture the enzymatically digested polypeptides for subsequent separation and analysis of the polypeptides.

[0081] The four-way valve 3 is connected to both the first six-way valve 2 and the second six-way valve 1, and the four-way valve 3 is also respectively connected to the intact protein separation column C4 and the polypeptide separation column C18a. The intact protein separation column C4 is used to separate intact proteins, while the polypeptide separation column C18a is used to separate the enzymatically digested polypeptides.

[0082] The trypsin protein column is arranged between the first six-way valve and the second six-way valve. Its working principle is that when the sample flows through the trypsin protein column, the trypsin in the trypsin protein column will enzymatically digest the protein in the sample and decompose the protein into polypeptides.

[0083] In the entire device, three important paths are formed. The first path consists of the sample inlet, the first six-way valve, and the quantitative loop. In actual work, by adjusting the path connection mode of the first six-way valve, the sample inlet is made to communicate with the quantitative loop, and the sample can flow from the sample inlet into the quantitative loop to achieve quantitative aspiration of the sample.

[0084] The second path consists of the sample inlet, the second six-way valve, the trypsin protein column, the capture column C18, the four-way valve, and the polypeptide separation column C18a. When the device switches to the working state of this path, the sample enters from the sample inlet and passes through the second six-way valve and the trypsin protein column in sequence, is enzymatically digested into polypeptides in the trypsin protein column, then the polypeptides flow through the capture column C18 and are captured, and finally, through the switching of the four-way valve, they enter the polypeptide separation column C18a for separation of the polypeptides.

[0085] The third route is composed of a mobile phase inlet, a first six-way valve, a sampling loop of the first six-way valve, a four-way valve, and a whole protein separation column C4. When this route is working, the mobile phase enters from the mobile phase inlet, passes through the first six-way valve and the sampling loop in sequence, and then through the switching of the four-way valve, enters the whole protein separation column C4, which is used for flushing, desalting and subsequent separation analysis of the whole protein.

[0086] Through the precise switching of each valve, the whole device realizes the spatio-temporal synchronous analysis of the whole protein and the enzymatically digested polypeptide, providing an efficient and accurate sample pretreatment and injection method for mass spectrometry analysis.

[0087] On the basis of the mass spectrometry injection integration device for spatio-temporal synchronous analysis of whole protein and enzymatically digested polypeptide described in claim 1, this embodiment further adds a first liquid phase pump and a second liquid phase pump.

[0088] The first liquid phase pump is connected to the sample inlet. Its function is to provide power for the transportation of the sample, and can precisely control the flow rate of the sample, so that the sample can stably enter the device from the sample inlet at the set rate. For example, in actual operation, the flow rate of the first liquid phase pump can be set to 0.4-0.6 uL / min, so as to ensure that the sample can be injected into the device at an appropriate speed, such as injected into the sampling loop of the first six-way valve, or enter the subsequent enzymatic digestion and polypeptide separation process through the second six-way valve.

[0089] The second liquid phase pump is connected to the mobile phase inlet. Its main function is to provide power for the transportation of the mobile phase and precisely control the flow rate of the mobile phase. The mobile phase plays a crucial role in the whole analysis process. It can drive the sample to be separated in each separation column. For example, in the whole protein separation process, the second liquid phase pump can transport the mobile phase from the mobile phase inlet at a flow rate of 2-4 uL / min, pass through the first six-way valve and the sampling loop, and then enter the whole protein separation column C4 through the four-way valve to flush, desalt and perform subsequent separation operations on the whole protein. In the polypeptide analysis process, the second liquid phase pump can transport the mobile phase at a specific flow rate and gradient. For example, at the initial stage of polypeptide analysis, the ratio of water to acetonitrile is set to 98%:2%, and the flow rate is set to 1.5 uL / min. Then, according to a specific mobile phase gradient, within 25-100 min, the proportion of acetonitrile gradually rises from 2% to 35%, so as to realize the step-by-step separation and elution of the polypeptide into the mass spectrometry detection.

[0090] In addition, the first six-way valve, the second six-way valve, and the four-way valve are connected by capillary lines to form a connected pipeline. The capillary line connecting pipeline has a small inner diameter, which can ensure the stable and accurate flow of liquid between each valve, reduce the diffusion and loss of samples and mobile phases during transmission, and ensure the analysis accuracy and stability of the entire device. Through the coordinated action of the first liquid-phase pump, the second liquid-phase pump, and the capillary line connecting pipeline, the mass spectrometry injection integration device for the spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides can operate more efficiently and accurately, providing reliable sample and mobile phase delivery guarantees for mass spectrometry analysis.

[0091] This embodiment describes a method for the spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides. First, the preparatory work is to inject a protein sample into a capillary quartz tube, and then dock the capillary quartz tube to the sample inlet.

[0092] Then enter the operation steps. In the first step, adjust the first six-way valve to form the first passage. At this time, start the first liquid-phase pump connected to the sample inlet and set the flow rate to 0.4 - 0.6 uL / min. This flow rate can ensure the stable and accurate injection of the sample. Under the action of the first liquid-phase pump, part of the sample in the capillary quartz tube is injected into the sample inlet and continues to flow until the sampling loop of the first six-way valve is filled with the sample. The purpose of this step is to accurately measure a certain volume of the sample for subsequent accurate analysis.

[0093] After that, perform the second operation step. Adjust the first six-way valve again and, at the same time, adjust the second six-way valve to form the second passage. At this time, the first liquid-phase pump continues to operate, and the remaining sample is injected into the trypsin column through the sample inlet. In the trypsin column, the proteins in the sample will start the enzymatic digestion reaction under the action of trypsin.

[0094] Immediately afterwards, perform the third operation step. Adjust the first six-way valve again and adjust the four-way valve to form the third passage. At this time, start the second liquid-phase pump connected to the mobile phase inlet and set the flow rate to 2 - 4 uL / min. The mobile phase enters from the mobile phase inlet, passes through the first six-way valve and the sampling loop in sequence, and then enters the intact protein separation column C4 through the four-way valve. In the intact protein separation column C4, the mobile phase flushes for 8 - 12 min at this flow rate for desalting operation to remove impurities such as salts in the sample for subsequent accurate analysis of intact proteins. At the same time, the remaining sample continues the on-line enzymatic digestion reaction on the trypsin column.

[0095] Subsequently, perform the fourth operation step. Adjust the first six-way valve again, adjust the second six-way valve, and adjust the four-way valve to form the second passage again. At this time, the first liquid-phase pump injects the remaining sample after enzymatic digestion into the polypeptide separation column C18a connected to the four-way valve. The polypeptide separation column C18a separates the enzymatically digested polypeptides according to the characteristics of the polypeptides.

[0096] Finally, the processed samples in the intact protein separation column C4 and the polypeptide capture column C18 are jointly delivered to a mass spectrometer for mass spectrometry data analysis. Through the analysis of the samples by the mass spectrometer, relevant information on intact proteins and enzymatically digested polypeptides can be obtained, thereby achieving the purpose of spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides. The entire analysis method precisely controls the switching of each valve and the flow rate of the liquid phase pump, and methodically completes a series of operations from sample injection to final mass spectrometry analysis, ensuring the accuracy and reliability of the analysis results.

[0097] The present invention relates to the fields of proteomics, mass spectrometry analysis technology, enzyme reactor technology, etc. Specifically, it relates to a mass spectrometry injection integration device for spatio-temporal synchronous analysis of intact proteins and enzymatically digested polypeptides, which is used to integrate bottom-up and top-down proteomics, synchronously and rapidly obtain more comprehensive protein structure information, and improve the efficiency of protein sample analysis.

[0098] Mass spectrometry-based proteomics has become a core tool for analyzing complex protein systems, showing irreplaceable advantages in fields such as high-throughput sequencing, precise quantification, dynamic monitoring of post-translational modifications, and analysis of protein interaction networks. The current mainstream strategies can be divided into two technical paths: "bottom-up" and "top-down", which form significant complements in terms of information dimension and detection efficiency.

[0099] The bottom-up strategy converts intact proteins into polypeptide fragments for mass spectrometry analysis through specific enzymatic digestion. With its ultra-high sensitivity (up to the amol level) and large-scale detection ability (more than 10,000 polypeptides can be identified in a single experiment), it has become the preferred solution for deep coverage of the proteome. However, due to the loss of high-order protein structure information during the enzymatic digestion process, it is difficult to resolve key biological problems such as the dynamic assembly of protein complexes and the precise typing of isomers. It is worth noting that although the deep learning-assisted polypeptide assembly algorithms reported in Nature Methods in 2023 (such as DeepNovo-DIA) have partially alleviated the sequence reconstruction error, the irreversible loss of structural information remains a fundamental limitation.

[0100] In contrast, the top-down strategy can completely retain key structural information such as post-translational modification combination patterns, subtype variations, and interaction interfaces by directly analyzing intact proteins and their complexes. The latest progress shows that, based on the technical breakthroughs of charge detection mass spectrometry (CDMS) and Orbitrap Astral, high-resolution characterization of protein complexes with a molecular weight greater than 200 kDa has been achieved. However, this strategy is still limited by technical bottlenecks such as narrow dynamic range (insufficient detection limit for low-abundance proteins) and low fragmentation efficiency (sequence coverage is mostly less than 60%), especially for the analysis of insoluble systems such as membrane proteins, which still faces significant challenges.

[0101] It is worth noting that both strategies are restricted by the lengthy sample pretreatment process: the bottom-up approach requires more than a dozen operations such as denaturation, reduction alkylation, and enzymatic digestion, while the top-down approach, although omitting the enzymatic digestion step, still requires repeated desalting and buffer replacement. Traditional offline processing methods not only lead to sample loss (especially for low-abundance targets), but also may introduce artificial modifications (such as methionine oxidation). Therefore, there is a need to develop an online analysis integration device for synchronous detection of intact proteins and enzymatically digested polypeptides in the same sample to quickly obtain protein information at the polypeptide level and the intact protein level.

[0102] The objective of the present invention is to provide an integrated analysis device for bottom-up and top-down proteomics based on liquid chromatography-mass spectrometry. Conventionally, mass spectrometry detection at the intact protein and polypeptide levels has independent sample pretreatment, enrichment, separation, and detection processes, which are not only time-consuming and laborious, but also require multiple offline operations such as protease digestion, desalting, and enrichment, easily leading to sample loss. In addition, single-level analysis of intact proteins and polypeptides will result in the loss of some important protein information. The present invention aims to develop an integrated mass spectrometry detection device for synchronous detection of intact proteins and polypeptides in the same sample, which can quickly perform online protein desalting, protein denaturation, protease digestion, polypeptide desalting, enrichment, and separation operations, and achieve synchronous detection of proteins from bottom-up and top-down.

[0103] The specific design concept is as follows:

[0104] Introduce the protein sample into the injection system of the integrated device. Under the action of the nanoliter liquid chromatography pump, by controlling the flow rate and residence time of the sample in the device, accurately allocate the injection volumes of the intact protein detection flow path and the polypeptide detection flow path of the integrated device; the sample entering the intact protein detection flow path is sequentially desalted, chromatographically separated, and mass spectrometrically detected in the capillary. At the same time, the protein sample entering the polypeptide detection flow path undergoes pretreatment processes such as denaturation, on-line protein digestion, and polypeptide desalting in the capillary. After the intact protein mass spectrometry detection and polypeptide pretreatment are completed synchronously, switch to the polypeptide detection flow path for chromatographic separation and mass spectrometry detection. Realize the synchronous acquisition of intact protein information and polypeptide information of the same protein sample in the same device.

[0105] Based on the technology of nano-liquid chromatography-mass spectrometry (nano-LC-MS), this invention constructs an integrated analysis device for synchronous detection of intact proteins and digested polypeptides from the same protein sample by integrating functional regions such as sample distribution, desalting and separation of intact proteins, denaturation of intact proteins, on-line digestion, desalting of polypeptides, separation of polypeptides, and mass spectrometry detection. It has the following differences compared with traditional proteomics research:

[0106] 1) Sample pretreatment scenario: In traditional methods, operations such as desalting, denaturation, and digestion of protein samples are off-line operations, which require complex processes such as sample transfer, reagent addition, and buffer exchange. All sample pretreatment processes in this invention are integrated in each functional region within the quartz capillary, and sample pretreatment is completed on-line.

[0107] 2) Sample utilization efficiency: Since all sample pretreatment processes in this research are completed within the capillary, after pretreatment, it can be directly subjected to mass spectrometry analysis without off-line operations such as sample transfer, reagent addition, and buffer exchange, greatly reducing the possibility of sample loss.

[0108] 3) Sample analysis speed: This invention uses self-developed technologies such as immobilized trypsin reactor, on-line desalting, and on-line protein denaturation, greatly shortening the detection speed of protein samples in complex matrices. The sample pretreatment only takes 20 minutes. However, traditional operations such as protein sample denaturation, digestion, desalting, and concentration take more than 24 hours.

[0109] 4) Protein information acquisition: This invention realizes synchronous detection of the same protein sample at the levels of intact proteins and digested polypeptides in the same set of devices, obtaining more comprehensive protein information compared with existing devices.

[0110] 5) Synchronous complementary analysis of sequence and higher-order structure information: The top-down protein analysis strategy adopted in this invention focuses on obtaining higher-order structure information such as the collision cross-sectional area of protein interactions and complexes, while the simultaneous bottom-up analysis can provide accurate qualitative and quantitative data support for protein sequence identification, effectively improving the reliability and analysis throughput of intact protein mass spectrometry analysis.

[0111] The present invention constructs an integrated mass spectrometry detection device for intact proteins and enzymatically digested polypeptides of the same protein sample, solving the problems of long detection time, complex operation and incomplete information acquisition for protein samples. Conventionally, bottom-up detection of protein samples in complex matrices requires sample pretreatment processes such as denaturation, enzymatic digestion, desalting and concentration, which greatly reduces the sample analysis throughput. In addition, current proteomics research is often single, only detecting intact proteins or enzymatically digested polypeptides, resulting in incomplete protein information acquisition. The present invention can solve the above problems by integrating bottom-up and top-down proteomics processes in one device.

[0112] The specific implementation scheme is as follows:

[0113] 1) Immobilized trypsin column packing:

[0114] Step 1: Take 200 uL of potassium silicate solution and mix it with 20 uL of formamide, and vortex mix (3000 rpm); cut a quartz capillary with an inner diameter of 200 um, an outer diameter of 370 um and a length of 15 cm, insert it into the potassium silicate and formamide mixture, suck the solution to a height of 1 cm inside the capillary, and place it in an oven at 100 °C and heat for 12 h to manufacture a column packing sieve plate.

[0115] Step 2: Dissolve the immobilized trypsin packing in 100 mM acetic acid solution at a concentration of about 1 mg / mL, and fill the chromatographic packing solution into the capillary with a sieve plate obtained in Step 1 using a capillary chromatographic column filling instrument, with a filling length of 5 cm.

[0116] Step 3: Use a micro-injection pump to inject ammonium bicarbonate solution (pH = 8) into the immobilized trypsin column at a flow rate of 2 uL / min in an environment at 4 °C to wash away the excess acetic acid in the column and balance the trypsin column for use in on-line enzymatic digestion experiments.

[0117] 2) Capillary chromatographic column packing and electrospray ionization needle manufacturing:

[0118] Step 4: Take a quartz capillary with an inner diameter of 100 um, an outer diameter of 365 um and a length of 20 cm. As in Step 1, suck the potassium silicate and formamide mixture to a height of 1 cm inside the capillary, and then place the capillary in an oven at 100 °C and heat for 12 h to obtain a quartz capillary with a sieve plate at one end for packing chromatographic separation packing.

[0119] Step 5: Dissolve 3 mg of C18 chromatographic packing (1.8 um, 100 A) in 600 uL of methanol solution and mix well. Use a capillary chromatographic column filling instrument to fill the chromatographic packing solution into the capillary with a sieve plate obtained in Step 9, with filling lengths of 5 cm and 15 cm respectively.

[0120] Step 6: Dissolve 3 mg of C4 chromatographic packing material (3 um, 300 A) in 600 uL of methanol solution and mix well. Use a capillary column filling instrument to fill the chromatographic packing material solution into the capillary with a sieve plate obtained in Step 9, and the filling length is 15 cm respectively.

[0121] Step 7: Take a quartz capillary with an inner diameter of 50 um, an outer diameter of 365 um, and a length of 10 cm. Use a laser pulling instrument to pull the capillary into an electrospray ionization needle, and the pulling parameters are as follows: Heat = 350, Fil = 1, Vel = 50, Pul = 30.

[0122] Step 8: Open the hole of the pulled needle by wet etching. Put the tip of the needle pulled in Step 11 into a 40% HF solution, and introduce nitrogen gas into the other end of the needle. The etching time is 5 min, and finally an electrospray ionization needle with an outer diameter of 365 um, an inner diameter of 50 um, and a tip aperture of 15 um is obtained.

[0123] 3) Integration of the bottom-up and top-down proteomics integrated analysis device:

[0124] Step 9: Connect the immobilized trypsin reactor obtained in Step 3, the capillary column obtained in Step 6, the electrospray ionization needle obtained in Step 8, the quartz capillary, the liquid phase pump, the six-port valve, the four-port valve, and the three-port valve according to Figure 1 (a) to complete the integration of the device.

[0125] Step 10: Balance and wash the C4 and C18 capillary columns with different ratios of acetonitrile and water. Thus, the integration of the bottom-up and top-down proteomics integrated analysis device is completed.

[0126] This technical solution provides a device and method for synchronous online detection of intact proteins and enzymatically digested polypeptides in the same protein sample. It has at least the following beneficial effects:

[0127] 1) Fast sample analysis speed: The integrated device for intact proteins and enzymatically digested polypeptides integrates processes such as online rapid enzymatic digestion, rapid desalting, and enrichment, shortening the time and spatial scale of sample pretreatment.

[0128] 2) Comprehensive protein information acquisition: It can obtain both intact protein information and polypeptide information after enzymatic digestion, effectively avoiding the loss of some important protein information, such as post-translational modifications.

[0129] 3) Avoid sample loss: All sample pretreatment operations are carried out inside the capillary, which can avoid losses caused by sample transfer and improve the utilization efficiency of the sample.

[0130] 4) Simple operation: During the processes of desalting, denaturation treatment, enzymatic digestion, enrichment, separation, and detection of the sample, it can be controlled by liquid phase and mass spectrometry methods (such as switching the position of the six-way valve, changing the mobile phase, and setting the mass spectrometry detection method, etc.), and the operation is relatively simple.

[0131] The integrated analysis device for intact proteins and enzymatically digested polypeptides of the same protein sample proposed by the present invention provides a new mass spectrometry injection device and a strategy for analyzing the higher-order structure of intact proteins for proteomics research based on mass spectrometry. The following are some key contributions and impacts that this invention may bring:

[0132] 1) Improve the analysis throughput and reliability of intact protein mass spectrometry technology

[0133] 2) Improve the ability to obtain protein higher-order structure information in trace samples

[0134] 3) Integrate the programmed multi-level detection of proteins in complex samples

[0135] Next, the present invention will be further elaborated in combination with three specific implementation examples.

[0136] Example 1: Evaluation of the online enzymatic digestion effect of protein samples

[0137] Online enzymatic digestion is an important link in the synchronous analysis device for intact proteins and enzymatically digested polypeptides of the same protein sample. Therefore, the online enzymatic digestion effect is evaluated first.

[0138] (1) Denaturation treatment of protein samples

[0139] Prepare cytochrome C at 1 mg / mL, hemoglobin at 1 mg / mL, and bovine serum albumin at 2 mg / mL respectively, with the solvent being phosphate buffer solution. Take 50 μL of each of the above three protein solutions, add 50 μL of 8 M urea and mix to denature the proteins.

[0140] (2) Online enzymatic digestion of protein samples

[0141] Add 900 μL of 25 mM ammonium bicarbonate solution to the denatured protein solution obtained in (1) for dilution, so that the urea concentration in the protein solution is lower than 1 M. Use a syringe to suck up the protein solution, and connect the outlet of the syringe solution to an immobilized trypsin column and a 5 cm long C18 capillary column in sequence. Under the push of the microsyringe, the protein sample passes through the immobilized trypsin column and the C18 chromatographic column in sequence, and the protein is captured in the 5 cm C18 chromatographic column after being enzymatically digested into polypeptides. The syringe flow rate is set at 0.5 μL / min, and the injection time is 20 min.

[0142] (3) Detection of enzymatically digested polypeptides

[0143] After the enzymatic digestion is completed, remove the 5-cm C18 capillary column that has captured the digested polypeptide. Connect one end of it to a liquid phase pump and flush the capillary column with a mobile phase of water:acetonitrile = 97%:3% at a flow rate of 3 μL / min for 10 min to remove the salts in the capillary column. After desalting, connect the other end of the capillary column to the mass spectrometer, and gradually increase the proportion of acetonitrile in the mobile phase to 50% within 10 min to quickly elute the polypeptide and identify it.

[0144] (4) Identification of protein samples

[0145] Import the data obtained in (3) into the PLGS software for database searching to identify the protein. The identification results are as Figure 2 shown. 10, 17, and 31 polypeptides were identified from cytochrome C, hemoglobin, and bovine serum albumin samples respectively, and the sequence coverages were 71.4%, 73.9%, and 48.4% respectively.

[0146] Example 2: Testing of the synchronous detection device for intact protein and digested polypeptide of a single protein sample

[0147] The synchronous detection device for intact protein and digested polypeptide of the same protein sample mainly consists of the following parts: ① 2 sets of liquid phase pumps; ② 2 six-port valves; ③ 1 four-port valve; ③ 2 three-ways; ④ polypeptide capture column (C18, 5 cm); ⑤ polypeptide separation column (C18, 15 cm); ⑥ intact protein separation column (C4, 15 cm); ⑦ micro syringe; ⑧ quantitative loop; ⑨ capillary line connection pipeline. Its operation mode is described in detail as follows:

[0148] Sample preparation:

[0149] Put the hemoglobin solution with a concentration of 0.4 mg / mL (containing 8 M Urea) into a 37 °C metal bath and incubate for 5 min, then dilute it with 25 mM ammonium bicarbonate solution to a Urea concentration of 1 M to completely denature the protein.

[0150] (2) Injection and distribution of hemoglobin sample:

[0151] Cut a 32-cm long, 200-μm inner diameter quartz capillary (volume 10 μL), fill the capillary with hemoglobin sample (0.05 mg / mL, 1 M urea), connect one end of the capillary to liquid phase pump 1, and the other side to the fifth position of the right six-port valve. The right six-port valve is at position A at this time, and its third and sixth positions are connected to a 7-μL quantitative loop. Liquid phase pump 1 injects the sample in the capillary into the quantitative loop of the right six-port valve at a flow rate of 0.5 μL / min. The mobile phase is 25 mM ammonium bicarbonate solution. After 15 min, switch the right six-port valve to position B, and liquid phase pump 1 pumps the remaining sample to the immobilized trypsin column device, as Figure 1 (a).

[0152] (2) Desalting, separation and mass spectrometry detection of intact protein

[0153] As Figure 1 (b) Adjust the right six-port valve to position B and the left six-port valve to position A. The liquid phase pump 2 uses a high proportion of aqueous phase as the mobile phase to pump the protein sample in the quantitative loop into the C4 capillary connected to the fourth position of the four-port valve at a flow rate of 3 uL / min and rinse for 10 min for desalting. Subsequently, the mass spectrometry starts data acquisition, and the proportion of the organic phase in the mobile phase of the liquid phase pump 2 is gradually increased. Its gradient setting is: within 0–25 min, the organic phase acetonitrile in the mobile phase gradually increases from 2% to 40%, and the intact protein sample is gradually separated and eluted, and the flow rate is set at 1.5 uL / min.

[0154] (3) On-line enzymatic digestion of protein sample

[0155] As Figure 1 (b) While performing desalting, separation and mass spectrometry detection of the intact protein, the liquid phase pump 1 still makes the sample flow to the trypsin column for on-line enzymatic digestion at a flow rate of 0.3 uL / min (with 25 mM ammonium bicarbonate as the mobile phase). Finally, the digested polypeptide is captured by a 5 cm C18 chromatographic column under the push of the liquid phase pump 1, and the solution flows out from the fourth outlet of the left six-port valve.

[0156] (4) Desalting, separation and mass spectrometry detection of polypeptide

[0157] As Figure 1 (c), when the on-line enzymatic digestion operation of the intact protein is completed, the mobile phase of pump 1 is changed to water, and the flow rate is set at 1 uL / min to rinse the trypsin column (about 60 min) to load the digested polypeptide sample onto the C18 capture column. Wait for the end of the intact protein analysis process. Adjust the right six-port valve to position A, the left six-port valve to position B, the four-port valve to position 2. The initial mobile phase ratio of the liquid phase pump 2 for polypeptide analysis is set as water:acetonitrile = 98%:2%, and the flow rate is 1.5 uL / min. Subsequently, the organic phase is gradually increased to gradually separate and elute the polypeptide into the mass spectrometry detection. The mobile phase gradient for polypeptide separation is: within 35 - 60 min, the proportion of acetonitrile gradually increases from 2% to 35%.

[0158] (5): Mass spectrometry data analysis

[0159] As Figure 3 As shown in the detection results of the intact protein, two chromatographic peaks of hemoglobin are detected. By calculating the protein molecular weight, it can be determined that they are the signals of the α subunit and β subunit of hemoglobin respectively; through the analysis of the digested polypeptide data ( Figure 4 ), a total of 18 polypeptides of hemoglobin are identified as specific enzymatically digested polypeptides of hemoglobin. The above results indicate that the integrated analysis device for intact protein and digested polypeptide can operate successfully and be applied to the rapid on-line detection of protein samples.

[0160] Example 3: Test of the Synchronous Detection Device for Intact Proteins and Enzymatically Digested Polypeptides in Bacterial Lysates

[0161] On the basis of Example 2, the present invention further tests the application effect of the integrated device in complex samples. In Example 3, the present invention uses the integrated device to synchronously obtain the information of intact proteins and enzymatically digested polypeptides in a sample of bacterial lysate (E. coli).

[0162] The composition of the synchronous detection device for intact proteins and enzymatically digested polypeptides in bacterial lysates is the same as that in Example 2, and its operation process is described in detail as follows:

[0163] Sample preparation:

[0164] The E. coli BL21 strain was placed in 150 mL of LB medium and cultured in a constant temperature shaker at 37 °C for 12 h. The final OD600 value of the bacteria was about 2.2. The bacterial solution was placed in a 50 mL centrifuge tube and centrifuged (5,000 rpm, 10 min) to obtain a precipitate, which was resuspended in 5 mL of binding buffer, and then the bacteria were ultrasonically disrupted under ice bath conditions. After the bacteria were disrupted, they were centrifuged (14,500 rpm, 20 min) to obtain the supernatant, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The protein concentration was measured by the BCA method, and the final protein concentration was set at 0.8 mg / mL using the binding buffer. Before loading, urea was added to the protein sample to a final concentration of 8 M, incubated in a 37 °C metal bath for 5 min, and then diluted to a urea concentration of 1 M with 25 mM ammonium bicarbonate solution. Subsequently, the sample was immediately introduced into the system.

[0165] The bacterial lysate sample was tested on the integrated device. The sample injection and distribution were the same as in Example 2

[0166] Desalting, separation, and mass spectrometry detection of intact proteins

[0167] The operation process of desalting and separation detection of intact proteins in bacterial lysates was basically the same as that in Example 2. Due to the more complex bacterial lysate sample, the separation gradient of its intact proteins was set as follows: within 0–80 min, the organic phase acetonitrile in the mobile phase gradually increased from 2% to 40%, and the intact protein sample was gradually separated and eluted, and the flow rate was set at 1.0 μL / min.

[0168] (4) On-line enzymatic digestion of protein samples

[0169] The on-line enzymatic digestion process of intact proteins in bacterial lysates was basically the same as that in Example 2. The main change was that the flow rate of the sample introduced into the trypsin column was changed to 0.1 μL / min to ensure sufficient on-line enzymatic digestion of various proteins.

[0170] (5) Desalting, separation, and mass spectrometry detection of polypeptides

[0171] The operation process of desalting, separating and mass spectrometry detection of polypeptides in bacterial lysate is basically the same as that in Example 2. Due to the more complex bacterial lysate sample, the separation gradient of intact proteins is set as follows: the organic phase acetonitrile in the mobile phase gradually increases from 2% to 35% within 0–100 min, and the polypeptides are gradually separated and eluted. The flow rate is set at 0.5 uL / min.

[0172] (6) Mass spectrometry data analysis

[0173] Bottom-up proteomics analysis and parsing were completed by using the PLGS search software configured by Waters Instruments. A total of 2,856 polypeptides, 413 proteins, and 386 proteomes were detected in the bacterial lysate (BL21). The molecular weight analysis of intact proteins was jointly completed by Intact Mass software and MaxEnt. The molecular weights of 171 proteins were analyzed, and their mass distribution ranges are as Figure 5 shown in c. Compared with bottom-up proteomics, the number of proteins identified by top-down proteomics is lower, mainly because the mass spectrometry has lower detection sensitivity for intact proteins compared with polypeptides, and the detected protein signals are limited. In addition, there are still some proteins detected by top-down proteomics but not detected by bottom-up proteomics ( Figure 5 d), which is mainly attributed to the following two aspects: ① Post-translational modification has occurred in the protein, and the molecular weight of the intact protein cannot match the detection results of the polypeptides after enzymatic digestion; ② Some protease cleavage sites are limited or there are no protease cleavage sites, so they cannot be detected by bottom-up proteomics.

[0174] Although the embodiments of the present invention have been disclosed as above, they are not limited to only the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated examples described herein.

Claims

1. An integrated mass spectrometry sampling device for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides, characterized in that: include: Sample inlet; a first six-way valve, the sample inlet being connected to the first six-way valve, and a quantitative loop being arranged in the first six-way valve; A second six-way valve, which is connected to the first six-way valve, the mobile phase inlet is connected to the second six-way valve, and the second six-way valve is provided with a capture column C18; A four-way valve, which is connected to both the first six-way valve and the second six-way valve, and the four-way valve is also connected to the intact protein separation column C4 and the polypeptide separation column C18a; and A pancreatic protein column, which is disposed between the first six-way valve and the second six-way valve; Among them, the following pathways are formed: The first passage is composed of a sample inlet, a first six-way valve, and a quantitative loop; The second passage consists of a sample inlet, a second six-way valve, a trypsin protein column, a capture column C18, a four-way valve, and a peptide separation column C18a; as well as The third passage is composed of a mobile phase inlet, a first six-way valve, a quantitative loop of the first six-way valve, a four-way valve, and a complete protein separation column C4.

2. The mass spectrometry sampling integrated device for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides as claimed in claim 1, characterized in that: Also includes: A first liquid phase pump, which is connected to the sample inlet; as well as A second liquid phase pump, which is connected to the mobile phase inlet; The first six-way valve, the second six-way valve, and the four-way valve are connected via a capillary line connection pipeline.

3. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides according to any one of claims 1 to 2, characterized in that: The following steps are involved: Inject the protein sample into the capillary quartz tube and connect it to the sample inlet; adjusting the first six-way valve so that a first passage is formed; Inject part of the sample in the capillary quartz tube into the sample inlet at a flow rate of 0.4-0.6uL / min until it reaches the quantitative loop of the first six-way valve; Afterwards, the first six-way valve is adjusted again, and the second six-way valve is adjusted so that a second passage is formed, and the remaining sample is injected into the trypsin column through the sample inlet; Afterwards, the first six-way valve and the four-way valve were adjusted again to form the third passage, and part of the sample in the quantitative loop was injected into the intact protein separation column C4 connected to the four-way valve through the mobile phase inlet, with a flow rate of 2-4uL / min, and the column was flushed for 8-12 minutes for desalting; at the same time, the remaining sample was subjected to online enzyme digestion on the trypsin column; Afterwards, the first six-way valve is opened again, the second six-way valve is adjusted, and the four-way valve is adjusted to form a second passage, and the polypeptide sample after enzyme digestion is injected into the polypeptide capture column C18; Finally, the processed samples in the intact protein separation column C4 and the peptide capture column C18 are introduced into the mass spectrometer for detection to obtain the data information of the intact protein and enzymatic peptides of the same sample.

4. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides as claimed in claim 3, characterized in that: The protein sample is Enterobacter BL21 strain protein, and its preparation method is as follows: The E. coli BL21 strain was placed in 120-160 mL of LB medium and cultured in a constant temperature shaker at 36-37°C for 11-13 h until the final OD600 value of the bacteria was 2.1-2.3; Transfer the cultured bacterial solution to a 50-60 mL centrifuge tube and centrifuge at 4000-6000 rpm for 8-12 min to obtain the precipitate; The above precipitate was resuspended in 5-6 mL of binding buffer, and the resuspended bacteria were ultrasonically disrupted under ice bath conditions; After the bacteria are broken, centrifuge at 14000-15000 rpm for 15-25 min to obtain the supernatant; The obtained supernatant was filtered through a 0.22 um water filter membrane; The protein concentration of the supernatant after membrane filtration was tested by the BCA method, and the final protein concentration was adjusted to 0.7-0.9 mg / mL using binding buffer; Before loading, add urea to the protein sample to a final concentration of 8-9 M, and then incubate the sample in a 37-38°C metal bath for 5-6 min; and After incubation, the sample was immediately diluted with 25 mM ammonium bicarbonate solution to a urea concentration of M to obtain the sample.

5. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides as claimed in claim 3, characterized in that: The enzymatic cleavage process includes: replacing the mobile phase of the first liquid phase pump connected to the sample inlet with water, setting the flow rate to 1-1.5uL / min, flushing the trypsin column for 50-70min, loading the polypeptide product hydrolyzed by the trypsin column onto the capture column C18, and waiting for the completion of the intact protein analysis process.

6. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides as claimed in claim 5, characterized in that: The second liquid phase pump connected to the mobile phase inlet is used to set the initial mobile phase ratio for peptide analysis so that the ratio of water to acetonitrile is 98%:2%; Then, peptides were separated according to a specific mobile phase gradient. Depending on the complexity of the sample, the proportion of acetonitrile was gradually increased from 2% to 35% within 25-100 minutes, and the peptides were gradually separated and eluted into mass spectrometry detection; depending on the complexity of the sample and the separation requirements, the flow rate was set to 0.5-1.5uL / min.

7. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides as claimed in claim 4, characterized in that: The second liquid phase pump connected to the mobile phase inlet is used to set the initial mobile phase ratio for peptide analysis so that the ratio of water to acetonitrile is 98%:2%; After that, complete separation is performed according to a specific mobile phase gradient. Depending on the complexity of the sample, the proportion of acetonitrile is gradually increased from 2% to 40% within 25-80 minutes, and the complete protein is gradually separated and eluted into mass spectrometry detection; depending on the complexity of the sample and the separation requirements, the flow rate is set to 1.0-1.5uL / min.

8. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides as claimed in claim 3, characterized in that: The protein sample is denatured, comprising the following steps: 1 mg / mL cytochrome C, 1 mg / mL hemoglobin, and 2 mg / mL bovine serum albumin were prepared respectively, and the solvents were all phosphate buffer; Take 50uL of each of the above three protein solutions, add 50uL of 8M urea and mix to denature the protein to obtain the final protein sample.

9. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides according to claim 8, characterized in that: The enzyme cleavage step comprises: Add 900uL of 25mM ammonium bicarbonate solution to the denatured protein solution to dilute it so that the urea concentration in the protein solution is less than 1M; The protein solution was siphoned with a quartz capillary tube with a length of 32 cm and an inner diameter of 200 μm, and both ends of the capillary tube filled with the protein solution were connected to the outlet of the first liquid phase pump, the trypsin fixed column and the capture column C18 respectively; Driven by the first liquid phase pump, the protein sample passes through the trypsin column and the capture column C18 in sequence, so that the protein sample is captured in the capture column C18 after being cleaved into polypeptides; The flow rate of the first liquid phase pump was set to 0.5uL / min, and the injection time was 12-16min.

10. The method for spatiotemporal synchronous analysis of intact proteins and enzymatic peptides as claimed in claim 3, characterized in that: Trypsin column packing methods include: Take 200uL of potassium silicate solution and mix it with 20uL of formamide, and vortex mix at 3000rpm; A quartz capillary with an inner diameter of 200um, an outer diameter of 370um, and a length of 15cm was cut and inserted into a mixture of potassium silicate and formamide. The solution was drawn to a height of 1cm in the capillary. The capillary was then placed in a 100°C oven and heated for 12h to prepare a column filling sieve plate. Dissolve the immobilized trypsin filler in 100 mM acetic acid solution to prepare a solution with a concentration of about 1 mg / mL; use a capillary chromatography column filling instrument to fill the above chromatography filler solution into the capillary with a sieve plate obtained in step 1, with a filling length of 5 cm; Using a microinjection pump, at 4°C, an ammonium bicarbonate solution with a pH of 8 was injected into the immobilized trypsin column with a flow rate set at 2uL / min to flush away excess acetic acid in the column and balance the trypsin column.

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