High-throughput complete glycopeptide full-automatic online enrichment and separation analysis method

Abstract

The invention discloses a high-throughput complete glycopeptide full-automatic online enrichment and separation analysis method which comprises the following steps: firstly, building a full-automatic online enrichment and separation analysis system capable of realizing synchronization of enrichment and separation analysis through switching of a ten-way valve; the full-automatic on-line enrichment and separation analysis system is adopted to enrich complete glycopeptides in a first-needle sample, then the full-automatic on-line enrichment and separation analysis system is adopted to enrich complete glycopeptides in a next-needle sample and separate and analyze complete glycopeptides in a previous-needle sample at the same time, and the above steps are repeated. The separation analysis of the complete glycopeptide in the last needle of sample is completed; the method has the advantages that the complete glycopeptide can be quickly, fully automatically and online enriched, separated and analyzed, the operation in the enrichment process is completely controllable, the problems of high sample loss rate, poor enrichment repeatability and low liquid chromatography separation degree can be greatly reduced, the time required by the complete glycopeptide enrichment and separation analysis steps is superposed, and the analysis accuracy is greatly improved. And the time required by the whole process can be obviously reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to a complete glycopeptide online enrichment and separation analysis method, and in particular to a high-throughput complete glycopeptide automatic online enrichment and separation analysis method. BACKGROUND

[0002] Protein glycosylation refers to the process of covalently linking sugar or glycan molecules to protein amino acid residues under the action of glycosyltransferase. A large number of existing studies have shown that glycosylation plays an important role in the nervous, immune, circulatory, reproductive, and digestive systems. Abnormal glycosylation is directly related to the occurrence of diseases such as tumors, infections, inflammation, immune diseases, and metabolic diseases. More than 80% of the FDA-approved clinical tumor protein markers can be glycosylated, including some sugar antigens. Therefore, in-depth study of protein glycosylation modification is of great significance in explaining the mechanism of life activities, screening biomarkers for diseases, and identifying drug targets. To achieve accurate identification of glycoproteins, nanoliter flow rate liquid chromatography-electrospray tandem mass spectrometry has become a fact standard for glycoproteomics and even proteomics research due to its high throughput, high sensitivity, and high resolution. The low chemical dose number of glycoproteins, the heterogeneity of glycosylation modification, and the low ionization efficiency of complete glycopeptides are the main obstacles in glycoproteomics research based on mass spectrometry. In order to improve the depth and reliability of protein glycosylation analysis, it is necessary to enrich complete glycopeptides in protein digests before mass spectrometry detection. Hydrophilic interaction chromatography, as a highly efficient and convenient glycopeptide enrichment method, is currently the mainstream glycopeptide enrichment method due to its high efficiency in glycopeptide enrichment and lack of bias in glycopeptide analysis. The complete glycopeptides obtained by enrichment from the sample are complex, and if directly detected by mass spectrometry, the accuracy and stability of glycopeptide identification are limited due to charge competition and ionization discrimination during electrospray ionization. Therefore, the complete glycopeptides after enrichment must be separated by appropriate methods to achieve mass spectrometry detection of different types of glycopeptides.

[0003] The existing enrichment of intact glycopeptides mainly uses pipette tips filled with hydrophilic packing, also known as hydrophilic interaction liquid chromatography tips. First, a low-polarity solution is used to dissolve the sample to obtain a sample solution, the sample solution is passed through the hydrophilic packing loaded in the hydrophilic interaction liquid chromatography tip, the sample is retained on the hydrophilic packing, then a large amount of loading buffer is passed through the hydrophilic interaction liquid chromatography tip to remove non-sugar peptides non-specifically adsorbed on the hydrophilic packing, and finally a strong-polarity buffer is used to flush the hydrophilic packing in the hydrophilic interaction liquid chromatography tip to elute the captured glycopeptides from the hydrophilic packing, thereby completing the manual enrichment of glycopeptides. Although the use of hydrophilic interaction liquid chromatography tips can achieve effective enrichment of intact glycopeptides, there are a large number of sample transfer steps in the process of enrichment of intact glycopeptides, and sample adsorption on the surface of containers and transfer tools will reduce the sensitivity of glycopeptide enrichment. In addition, this method relies on manual and semi-automatic operation, and due to human intervention, the reproducibility of intact glycopeptide enrichment depends largely on the experience and level of the operator.

[0004] High-efficiency separation of intact glycopeptides before detection can significantly reduce the complexity of the intact glycopeptide detection process, thereby improving the depth of intact glycopeptide identification. Currently, nanoscale reversed-phase liquid chromatography is mainly used for separation and analysis of intact glycopeptides. In order to improve the speed of sample analysis, a method of coupling a trapping column and an analytical column is usually used. A low-concentration sample solution first flows through the trapping column at a high flow rate, and the analyte is concentrated and fixed on the trapping column. Then, the trapping column and the analytical column are connected, and a chromatographic gradient with a decreasing organic phase ratio is used to elute the analyte from the trapping column and separate it through the analytical column before entering the mass spectrometer for detection. Although this method of coupling chromatographic columns can improve the throughput of sample analysis, the spectral band broadening in the trapping column during sample trapping will continue to the sample separation process in the analytical column, which will affect the separation degree of liquid chromatography. The use of short-chain reversed-phase chromatographic packing in the trapping column and long-chain chromatographic packing in the analytical column can elute the analyte from the trapping column during sample separation and achieve secondary concentration at the head of the analytical column. This method can significantly compress the chromatographic peak width of the analyte and improve the separation degree of liquid chromatography.

[0005] It is worth noting that the enrichment and liquid chromatography separation and analysis of intact glycopeptides are currently carried out as two separate experimental parts. A large amount of time is spent on the enrichment step of intact glycopeptides before using liquid chromatography for separation and analysis of intact glycopeptides, which limits the throughput of glycopeptide identification in samples.

[0006] In summary, using machinery instead of manual labor can achieve stable reproducibility of whole glycopeptide enrichment operations; combining different separation packing materials can improve the liquid phase separation of whole glycopeptides; and superimposing the time required for whole glycopeptide enrichment and separation steps can significantly reduce the overall analytical time. Therefore, there is an urgent need to develop a high-throughput, fully automated online enrichment and separation method for whole glycopeptides. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a high-throughput fully automated online enrichment and separation method for complete glycopeptides that can rapidly, fully automatically, and online, with complete controllability during the enrichment process. This method can greatly reduce the problems of high sample loss rate, poor enrichment repeatability, and low liquid chromatography resolution caused by manual operation. Furthermore, by combining the time required for the enrichment and separation analysis steps of complete glycopeptides, the overall process time can be significantly reduced.

[0008] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a high-throughput fully automated online enrichment and separation analysis method for complete glycopeptides. First, a fully automated online enrichment and separation analysis system capable of simultaneous enrichment and separation analysis via a ten-way valve is built. Then, the fully automated online enrichment and separation analysis system is used to enrich complete glycopeptides in the first sample. Then, the fully automated online enrichment and separation analysis system is used to enrich complete glycopeptides in the next sample while simultaneously performing separation analysis of complete glycopeptides in the previous sample. This process is repeated until the separation analysis of complete glycopeptides in the last sample is completed.

[0009] Compared with existing technologies, the advantages of this invention lie in the construction of a fully automated online enrichment and separation analysis system. After enriching intact glycopeptides in the first sample, this system switches via a ten-way valve to simultaneously enrich intact glycopeptides in the next sample and separate and analyze intact glycopeptides from the previous sample. This allows for rapid, fully automated, and online enrichment and separation analysis of intact glycopeptides, with complete controllability during the enrichment process. This significantly reduces the problems of high sample loss rate, poor enrichment repeatability, and low liquid chromatography resolution caused by manual operation. Furthermore, the time required for the complete glycopeptide enrichment and separation analysis steps is superimposed, which can significantly reduce the overall process time.

[0010] Furthermore, the fully automated online enrichment and separation analysis system includes a column oven, two 10-port valves, a high-pressure gas cylinder, three waste liquid tubes, two waste liquid containers, nine plugs, a loading pump, a nanoliter chromatography pump, a mass spectrometry electrospray ionization source, a mass spectrometer, a hydrophilic interaction liquid chromatography column (i.e., a hydrophilic column), a reversed-phase chromatography trapping column (i.e., a trapping column), and a reversed-phase chromatography analytical column (i.e., an analytical column); the loading pump is a plunger pump, the nanoliter chromatography pump is a binary plunger pump, and each 10-port valve has ten ports; the two 10-port valves are respectively installed on the column oven and are referred to as the first 10-port valve and the second 10-port valve; the nine plugs are referred to as the first plug to the ninth plug; the three waste liquid tubes are referred to as the first waste liquid tube, the second waste liquid tube, and the third waste liquid tube; the two waste liquid containers are referred to as the first waste liquid container and the second waste liquid container; the outlet of the high-pressure gas cylinder... The first, second, third, fourth, fifth, and sixth plugs, the first waste liquid inlet, the loading pump outlet, and the hydrophilic column inlet are installed in a clockwise order at the ten ports of the first ten-way valve; controlling the valve position of the first ten-way valve enables the loading pump outlet and the hydrophilic column inlet to communicate. The seventh, eighth, and ninth plugs, the second waste liquid inlet, the hydrophilic column outlet, the trapping column inlet, the nanoliter chromatography pump outlet, the analytical column inlet, the trapping column outlet, and the third waste liquid inlet are installed in a clockwise order at the ten ports of the second ten-way valve; controlling the valve position of the second ten-way valve enables the trapping column outlet and the analytical column inlet to communicate. The analytical column outlet is connected to the mass spectrometry electrospray ionization source, the mass spectrometry electrospray ionization source is connected to the mass spectrometer, the first waste liquid outlet is connected to the first waste liquid tank, and the second and third waste liquid outlets are respectively connected to the second waste liquid tank.

[0011] Furthermore, the hydrophilic column, trapping column, and analytical column are prepared as follows: Three capillaries, each open at one end and with a porous sieve plate at the other, are connected to a high-pressure chamber. A suspension of hydrophilic packing material in methanol is added to the high-pressure chamber connected to the first capillary and sealed. A suspension of reversed-phase chromatography packing material in methanol is added to the high-pressure chamber connected to the second capillary and sealed. A suspension of reversed-phase chromatography packing material in methanol is added to the high-pressure chamber connected to the third capillary and sealed. Each high-pressure chamber is then immersed in water, and a plunger pump is used to pump pressure into each high-pressure chamber. For methanol at 800 bar, a high-pressure homogenization packing method is used to fill the first capillary with one open end and a porous sieve plate at the other end using a high-pressure chamber. Then, reversed-phase chromatography packing material is filled into the second capillary with one open end and a porous sieve plate at the other end, and finally into the third capillary with one open end and a porous sieve plate at the other end. Each capillary with one open end and a porous sieve plate at the other end is then vibrated using an ultrasonic instrument to ensure uniform packing. At this point, the first capillary with one open end and a porous sieve plate at the other end filled with hydrophilic packing material forms a hydrophilic column, the second capillary with one open end and a porous sieve plate at the other end filled with reversed-phase chromatography packing material forms a trapping column, and the third capillary with one open end and a porous sieve plate at the other end filled with reversed-phase chromatography packing material forms an analytical column.

[0012] Furthermore, the capillary tube with one open end and a porous sieve plate at the other end is prepared by the following steps:

[0013] Step A1: Select a suitable capillary tube according to the quality of the sample to be processed. The capillary tube can be a quartz capillary tube or a stainless steel capillary tube, and cut the capillary tube to the required length for later use.

[0014] Step A2: Mix formamide and potassium silicate solution at a volume ratio of 1:50 to 50:1, shake thoroughly until completely mixed, centrifuge and retain the supernatant;

[0015] Step A3: Insert one end of the prepared capillary tube into the supernatant in a direction perpendicular to the supernatant until a portion of the capillary tube, which is 0.5 to 1.0 cm long, is completely immersed in the supernatant. At this time, the supernatant will be drawn into the capillary tube under the action of capillary, causing the liquid level inside the capillary tube to rise. Maintain this state until the liquid level of the supernatant inside and outside the capillary tube stabilizes and no longer changes.

[0016] Step A4: Take out the soaked capillary tube vertically, keeping the capillary tube vertical, wipe off the supernatant adhering to the outer wall of the capillary tube, and then place the capillary tube vertically in an oven for heating, so that the supernatant is fully solidified at one end of the capillary tube to form a porous sieve plate. At this time, a capillary tube with one end open and the other end having a porous sieve plate is obtained.

[0017] Furthermore, the hydrophilic filler is any one or a mixture of two of the following: hydrophilic interaction fillers and zwitterionic hydrophilic interaction fillers.

[0018] Furthermore, the reverse chromatography packing material is any one or a mixture of at least two of C4 packing material, C8 packing material, C10 packing material, C18 packing material, and C30 packing material.

[0019] Furthermore, the alkyl chain length of the reversed-phase chromatography packing material in the trapping column is less than or equal to the alkyl chain length of the reversed-phase chromatography packing material in the analytical column.

[0020] Furthermore, the specific process for enriching intact glycopeptides in the first sample using this fully automated online enrichment and separation analysis system is as follows:

[0021] S2.1 Control the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column; control the position of the second ten-way valve to connect the outlet of the trapping column and the inlet of the analysis column.

[0022] S2.2 Dissolve the preset dose of the first injection sample (i.e., protein hydrolysate) in the loading buffer to obtain the first injection sample solution. The concentration of the first injection sample in the first injection sample solution is 1 nanogram per microliter to 1 milligram per microliter.

[0023] S2.3 First, use a loading pump to load the first sample solution onto the hydrophilic column at a flow rate of 1-500 μL per minute. Then, use the loading pump to deliver 1-500 times the column volume of loading buffer to flush the hydrophilic column, eluting the non-glycopeptides in the first sample solution onto the hydrophilic column. Next, use a nano-chromatographic pump to pump the mobile phase solvent with an organic phase ratio that increases linearly over time into the trapping and analytical columns until the organic phase ratio in the mobile phase solvent reaches the preset maximum value. Clean the trapping and analytical columns before enrichment and separation.

[0024] S2.4 Switch the position of the first ten-way valve to connect the outlet of the high-pressure gas cylinder with the inlet of the hydrophilic column, while keeping the position of the second ten-way valve unchanged.

[0025] S2.5. Use a loading pump to deliver 1 μL to 10 mL of elution buffer to flush the loading pump. Use a high-pressure gas cylinder to introduce high-pressure gas at a pressure of 0.1-10 MPa into the hydrophilic column and maintain this pressure for 0.1-300 minutes. Simultaneously, use a nanoliter chromatography pump to pump 1 nanoliter to 100 μL of equilibration buffer sequentially through the trapping column and the analytical column to equilibrate them.

[0026] S2.6 Switch the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column, and switch the position of the second ten-way valve to connect the outlet of the hydrophilic column and the inlet of the collection column.

[0027] S2.7 Using a loading pump, continuously pump 1-200 times the column volume of elution buffer through the hydrophilic column and the trapping column sequentially to elute the glycopeptides retained on the hydrophilic column and transfer them to the trapping column. The enrichment of intact glycopeptides in the first sample is completed. At the same time, use a nano-chromatographic pump to pump 1 nanoliter-100 microliters of equilibration buffer through the analytical column to make the analytical column equilibrate in the equilibration buffer.

[0028] S2.8 Control the position of the 20th valve so that the outlet of the collection column and the inlet of the analysis column are connected, while the position of the 10th valve remains unchanged;

[0029] S2.9. Use a loading pump to continuously pump 1-200 times the column volume of loading buffer to the hydrophilic column to bring it to equilibrium; at the same time, use a nano-chromatographic pump to pump 1 nanoliter to 100 microliters of equilibration buffer sequentially through the trapping column and the analytical column to bring them to equilibrium.

[0030] Furthermore, the specific process of using this fully automated online enrichment and separation analysis system to enrich intact glycopeptides in the next sample while simultaneously performing separation analysis of intact glycopeptides in the previous sample is as follows:

[0031] S3.1 Control the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column; control the position of the second ten-way valve to connect the outlet of the trapping column and the inlet of the analysis column.

[0032] S3.2 Dissolve the next dose of sample (i.e., protein hydrolysate) in loading buffer to obtain a solution with a protein hydrolysate concentration of 1 ng / µL to 1 mg / µL as the next dose sample solution.

[0033] S3.3. Use a loading pump to load the next injection sample solution onto the hydrophilic column at a flow rate of 1-500 μL / min. Then, use the loading pump to deliver 1-500 times the column volume of loading buffer to flush the hydrophilic column, eluting the non-glycopeptides from the previous injection sample solution onto the hydrophilic column. Simultaneously, a nano-chromatographic pump pumps a mobile phase solvent with an organic phase ratio that increases linearly or piecewise linearly over time into the trap column and the analytical column until the organic phase ratio in the mobile phase solvent reaches the preset maximum value. During this process, the intact glycopeptides from the previous injection sample stored on the trap column are separated by the analytical column and then enter the mass spectrometry electrospray ionization source. The mass spectrometry electrospray ionization source ionizes the intact glycopeptides from the previous injection sample and sends them to the mass spectrometer. The mass spectrometer outputs the mass spectrometry analysis data of the intact glycopeptides from the previous injection sample.

[0034] S3.4 Switch the position of the first ten-way valve to connect the outlet of the high-pressure gas cylinder with the inlet of the hydrophilic column, while keeping the position of the second ten-way valve unchanged.

[0035] S3.5. Use a loading pump to deliver 1 μL to 10 mL of elution buffer to flush the loading pump and its connecting tubing; use a high-pressure gas cylinder to introduce high-pressure gas at a pressure of 0.1-10 MPa into the hydrophilic column and maintain this pressure for 0.1-300 minutes; simultaneously, use a nanoliter chromatography pump to pump 1 nanoliter to 100 μL of equilibration buffer sequentially through the trapping column and the analytical column to equilibrate them.

[0036] S3.6 Switch the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column, and switch the position of the second ten-way valve to connect the outlet of the hydrophilic column and the inlet of the collection column.

[0037] S3.7 Using a loading pump, continuously pump 1-200 times the column volume of elution buffer through the hydrophilic column and the trapping column sequentially to elute the glycopeptides retained on the hydrophilic column and transfer them to the trapping column. The enrichment of intact glycopeptides in the next sample is completed. At the same time, use a nano-chromatographic pump to pump 1 nanoliter-100 microliters of equilibration buffer through the analytical column to make the analytical column equilibrate in the equilibration buffer.

[0038] S3.8 Control the position of the 20th valve so that the outlet of the collection column and the inlet of the analysis column are connected, while the position of the 10th valve remains unchanged;

[0039] S3.9. Use a loading pump to continuously pump 1-200 times the column volume of loading buffer to the hydrophilic column to bring it to equilibrium. At the same time, use a nano-chromatographic pump to pump 1 nanoliter to 100 microliters of equilibration buffer sequentially through the trapping column and the analytical column to bring them to equilibrium.

[0040] Furthermore, in the loading buffer, the organic solvent accounts for 50%-99% of the volume, the organic acid accounts for 0%-10% of the volume, and the remainder is water; in the elution buffer, the organic solvent accounts for 0%-15% of the volume, the organic acid accounts for 0%-10% of the volume, and the remainder is water; in the equilibration buffer, the organic solvent accounts for 0%-15% of the volume, the organic acid accounts for 0%-10% of the volume, and the remainder is water; the mobile phase solvent at its lowest organic phase volume percentage is: 0.1%-15% organic phase, 0%-10% organic acid, and the remainder is water; the mobile phase solvent at its highest organic phase volume percentage is: 30%-99% organic phase, 0%-10% organic acid, and the remainder is water.

[0041] Furthermore, the high-pressure gas is any one or a mixture of any of the following: air, nitrogen, oxygen, hydrogen, helium, argon, and carbon dioxide.

[0042] Furthermore, the organic solvent is any one or a mixture of any of the following: acetonitrile, methanol, ethanol, isopropanol, tetrahydrofuran, dichloromethane, and acetone.

[0043] Furthermore, the organic acid is any one or a mixture of any of formic acid, acetic acid, and trifluoroacetic acid. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the fully automated online enrichment and separation analysis system in the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention.

[0045] Figure 2 This is a schematic diagram of the packing device for the hydrophilic column, trapping column, and analytical column in the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention.

[0046] Figure 3 The mass spectrometry total ion chromatogram of a single sample obtained in Example 4 of the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention;

[0047] Figure 4 The mass spectrometry total ion chromatogram of a single sample obtained in Example 5 of the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention;

[0048] Figure 5 The mass spectrometry total ion chromatogram of a single sample obtained in Example 6 of the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention;

[0049] Figure 6This is a mass spectrometry total ion chromatogram of a single sample obtained in Example 7 of the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention.

[0050] Figure 7 The mass spectrometry total ion chromatogram of a single sample obtained in Example 8 of the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention;

[0051] Figure 8 This is a mass spectrometry total ion chromatogram of a sample obtained in Example 9 of the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0053] Example 1: A high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides. First, a fully automated online enrichment and separation analysis system capable of simultaneous enrichment and separation analysis via a ten-way valve is built. Then, the fully automated online enrichment and separation analysis system is used to enrich intact glycopeptides in the first sample. Then, the fully automated online enrichment and separation analysis system is used to enrich intact glycopeptides in the next sample while simultaneously performing separation analysis of intact glycopeptides in the previous sample. This process is repeated until the separation analysis of intact glycopeptides in the last sample is completed.

[0054] In this embodiment, by switching via a ten-way valve, the fully automated online enrichment and separation analysis system can simultaneously enrich intact glycopeptides in the next sample and separate and analyze intact glycopeptides in the previous sample. Thus, the fully automated online enrichment and separation analysis system can rapidly, automatically, and online enrich and separate intact glycopeptides, and the operation during the enrichment process is completely controllable. This can greatly reduce the problems of high sample loss rate, poor enrichment repeatability, and low liquid chromatography resolution caused by manual operation. At the same time, the time required for the complete glycopeptide enrichment and separation analysis steps is superimposed, which can significantly reduce the time required for the entire process.

[0055] Example 2: This example is basically the same as Example 1, except that: in this example, as Figure 1As shown, the fully automated online enrichment and separation analysis system includes a column oven 1, two 10-port valves, a high-pressure gas cylinder 2, three waste liquid tubes 3, two waste liquid tanks, nine plugs, a loading pump 4, a nanoliter chromatography pump 5, a mass spectrometry electrospray ionization source 6, a mass spectrometer, a hydrophilic interaction liquid chromatography column (i.e., hydrophilic column 7), a reversed-phase chromatography trapping column (i.e., trapping column 8), and a reversed-phase chromatography analytical column (i.e., analytical column 9). The loading pump 4 is a plunger pump, and the nanoliter chromatography pump 5 is a binary plunger pump. Each 10-port valve has ten ports. The two 10-port valves are installed on the column oven 1 and are respectively called the first 10-port valve 10 and the second 11. The nine plugs are respectively called the first plug to the ninth plug. The three waste liquid tubes 3 are respectively called the first waste liquid tube 3, the second waste liquid tube 3, and the third waste liquid tube 3. The two waste liquid tanks are respectively called the first waste liquid tank and the second waste liquid tank. The second waste liquid tank 12; the outlet of the high-pressure gas cylinder 2, the first plug, the second plug, the third plug, the fourth plug, the fifth plug, the sixth plug, the inlet of the first waste liquid pipe 3, the outlet of the loading pump 4, and the inlet of the hydrophilic column 7 are installed in a clockwise order at the ten ports of the first ten-way valve 10; controlling the valve position of the first ten-way valve 10 enables the outlet of the loading pump 4 and the inlet of the hydrophilic column 7 to communicate; the seventh plug, the eighth plug, the ninth plug, the inlet of the second waste liquid pipe 3, the outlet of the hydrophilic column 7, the inlet of the trapping column 8, the outlet of the nano-chromatographic pump 5, the inlet of the analytical column 9, the outlet of the trapping column 8, and the inlet of the third waste liquid pipe 3 are installed in a clockwise order at the ten ports of the second ten-way valve 11; controlling the valve position of the second ten-way valve 11 enables the outlet of the trapping column 8 and the inlet of the analytical column 9 to communicate; the outlet of the analytical column 9 is connected to the mass spectrometry electrospray ionization source 6, and the mass spectrometry electrospray ionization source 6 is connected to the mass spectrometer. The outlet of the first waste liquid pipe 3 is connected to the first waste liquid tank, and the outlets of the second waste liquid pipe 3 and the third waste liquid pipe 3 are respectively connected to the second waste liquid tank 12.

[0056] Example 3: This example is basically the same as Example 2, except that: in this example, as Figure 2As shown, the hydrophilic column 7, the trapping column 8, and the analytical column 9 are prepared as follows: Three capillary tubes 14, each open at one end and with a porous sieve plate 13 at the other end, are prepared. The opening of each capillary tube 14 is connected to a high-pressure chamber 15. A suspension of hydrophilic packing material in methanol is added to the high-pressure chamber connected to the first capillary tube 14 and sealed. A suspension of C8 packing material in methanol is added to the high-pressure chamber connected to the second capillary tube 14 and sealed. A suspension of C18 packing material in methanol is added to the high-pressure chamber connected to the third capillary tube 14 and sealed. Each high-pressure chamber 15 is immersed in water, and a plunger pump 18 is used to pump water into each high-pressure chamber 15 at a pressure of [pressure value missing]. Methanol at 800 bar is used to fill a capillary tube 14 with a high-pressure homogenization method. Hydrophilic packing material is filled into the first capillary tube 14, which is open at one end and has a porous sieve plate 13 at the other end. C8 packing material is filled into the second capillary tube 14, which is open at one end and has a porous sieve plate 13 at the other end. C18 packing material is filled into the third capillary tube 14, which is open at one end and has a porous sieve plate 13 at the other end. Each capillary tube 14 is then vibrated by an ultrasonic instrument 19 to ensure uniform packing. At this point, the first capillary tube 14 filled with hydrophilic packing material forms a hydrophilic column 7, the second capillary tube 14 filled with C8 packing material forms a trapping column 8, and the third capillary tube 14 filled with C18 packing material forms an analytical column 9.

[0057] In this embodiment, the combination of the trapping column 8 packed with C8 packing and the analytical column 9 packed with C18 packing can achieve secondary concentration of the analyte during the separation process, which significantly improves the liquid chromatography resolution of the analyte.

[0058] Example 4: This example is basically the same as Example 3, except that: in this example, the capillary tube with one open end and a porous sieve plate at the other end is prepared by the following steps:

[0059] Step A1: Select a suitable capillary tube according to the quality of the sample to be processed. The capillary tube can be a quartz capillary tube or a stainless steel capillary tube, and cut the capillary tube to the required length for later use.

[0060] Step A2: Mix formamide and potassium silicate solution in a volume ratio of 1:5, shake thoroughly until completely combined, centrifuge and retain the supernatant;

[0061] Step A3: Insert one end of the prepared capillary tube into the supernatant in a direction perpendicular to the supernatant until a portion of the capillary tube with a length of 0.5 cm is completely immersed in the supernatant. At this time, under the action of capillary action, the supernatant will be drawn into the capillary tube, causing the liquid level inside the capillary tube to rise. Maintain this state until the liquid level height of the supernatant inside and outside the capillary tube stabilizes and no longer changes.

[0062] Step A4: Take out the soaked capillary tube vertically, keeping the capillary tube vertical, wipe off the supernatant adhering to the outer wall of the capillary tube, and then place the capillary tube vertically in a 120℃ oven for heating, so that the supernatant is fully solidified at one end of the capillary tube to form a porous sieve plate. At this time, a capillary tube with one end open and the other end having a porous sieve plate is obtained.

[0063] In this embodiment, when preparing a capillary tube for the hydrophilic column 7 with one end open and the other end having a porous sieve plate, a quartz capillary tube with an inner diameter of 530 micrometers is selected, and a capillary tube with a length of 17 cm is cut for later use; when preparing a capillary tube for the collection column 8 with one end open and the other end having a porous sieve plate, a quartz capillary tube with an inner diameter of 75 micrometers is selected, and a capillary tube with a length of 10 cm is cut for later use; when preparing a capillary tube for the analytical column 9 with one end open and the other end having a porous sieve plate, a quartz capillary tube with an inner diameter of 75 micrometers is selected, and a capillary tube with a length of 75 cm is cut for later use.

[0064] In this embodiment, the specific process of enriching intact glycopeptides in the first sample using the fully automated online enrichment and separation analysis system is as follows:

[0065] S2.1 Control the position of the first ten-way valve 10 so that the outlet of the loading pump 4 and the inlet of the hydrophilic column 7 are connected; control the position of the second ten-way valve 11 so that the outlet of the collection column 8 and the inlet of the analysis column 9 are connected.

[0066] S2.2 Dissolve 1 microgram of the first injection sample (i.e., mouse brain protein hydrolysate) in the loading buffer to obtain the first injection sample solution. The concentration of the first injection sample in the first injection sample solution is 2 micrograms per microliter.

[0067] S2.3 First, use loading pump 4 to load the first sample solution onto the hydrophilic column 7 at a flow rate of 10 μL per minute. Then, use loading pump 4 to deliver 100 times the column volume of loading buffer to flush the hydrophilic column 7, eluting the non-glycopeptides in the first sample solution on the hydrophilic column 7. Then, use nano-chromatographic pump 5 to pump the mobile phase solvent with an organic phase ratio that increases linearly with time into the trapping column 8 and the analytical column 9 until the organic phase ratio in the mobile phase solvent reaches the preset maximum value. Clean the trapping column 8 and the analytical column 9 before enrichment and separation.

[0068] S2.4 Switch the position of the first ten-way valve 10 so that the outlet of the high-pressure gas cylinder 2 is connected to the inlet of the hydrophilic column 7, while the position of the second ten-way valve 11 remains unchanged.

[0069] S2.5. Use loading pump 4 to deliver 1 ml of elution buffer to flush loading pump 4. Use high-pressure gas cylinder 2 to introduce nitrogen gas at a pressure of 0.6 MPa into hydrophilic column 7 and maintain it for 20 minutes. At the same time, use nano-chromatographic pump 5 to pump 6 μL of equilibration buffer through the trapping column 8 and the analytical column 9 in sequence to equilibrate the trapping column 8 and the analytical column 9.

[0070] S2.6 Switch the first ten-way valve 10 to the valve position so that the outlet of the loading pump 4 and the inlet of the hydrophilic column 7 are connected. Switch the second ten-way valve 11 to the valve position so that the outlet of the hydrophilic column 7 and the inlet of the collecting column 8 are connected.

[0071] S2.7 Using loading pump 4, continuously pump elution buffer at a volume 3 times that of hydrophilic column 7 through hydrophilic column 7 and trapping column 8 in sequence to elute the glycopeptides retained on hydrophilic column 7 and transfer them to trapping column 8. The enrichment of intact glycopeptides in the first sample is completed. At the same time, use nano-chromatographic pump 5 to pump 900 nanoliters of equilibration buffer through analytical column 9 to make analytical column 9 equilibrate in equilibration buffer.

[0072] S2.8 Control the position of the second ten-way valve 11 so that the outlet of the collection column 8 and the inlet of the analysis column 9 are connected, while the position of the first ten-way valve 10 remains unchanged;

[0073] S2.9. Using loading pump 4, continuously pump 30 times the column volume of loading buffer to hydrophilic column 7 to bring hydrophilic column 7 to equilibrium; at the same time, using nano-chromatographic pump 5, pump microliters of equilibration buffer 6 sequentially through trap column 8 and analytical column 9 to bring trap column 8 and analytical column 9 to equilibrium.

[0074] In this embodiment, the specific process of using the fully automated online enrichment and separation analysis system to enrich intact glycopeptides in the next sample while simultaneously performing separation analysis of intact glycopeptides in the previous sample is as follows:

[0075] S3.1 Control the position of the first ten-way valve 10 so that the outlet of the loading pump 4 and the inlet of the hydrophilic column 7 are connected; control the position of the second ten-way valve 11 so that the outlet of the collection column 8 and the inlet of the analysis column 9 are connected.

[0076] S3.2 Dissolve the next dose of sample (i.e., mouse brain protein hydrolysate) in loading buffer to obtain a solution with a protein hydrolysate concentration of 2 micrograms per microliter as the next dose sample solution.

[0077] S3.3. Using loading pump 4, the next injection sample solution is loaded onto hydrophilic column 7 at a flow rate of 10 μL / min. Then, loading pump 4 is used to deliver 100 times the column volume of loading buffer to flush hydrophilic column 7, eluting non-glycopeptides from the previous injection sample solution onto and off hydrophilic column 7. Simultaneously, nano-chromatographic pump 5 pumps a mobile phase solvent with an organic phase ratio that increases linearly or piecewise linearly over time into trap column 8 and analytical column 9 until the organic phase ratio in the mobile phase solvent reaches the preset maximum value. During this process, the intact glycopeptides from the previous injection sample stored on trap column 8 are separated by analytical column 9 and enter mass spectrometry electrospray ionization source 6. Mass spectrometry electrospray ionization source 6 ionizes the intact glycopeptides from the previous injection sample and sends them to the mass spectrometer. The mass spectrometer outputs the mass spectrometry analysis data of the intact glycopeptides from the previous injection sample.

[0078] S3.4 Switch the position of the first ten-way valve 10 so that the outlet of the high-pressure gas cylinder 2 is connected to the inlet of the hydrophilic column 7, while the position of the second ten-way valve 11 remains unchanged.

[0079] S3.5. Use loading pump 4 to deliver 1 ml of elution buffer to flush loading pump 4 and its connecting tubing; use high-pressure gas cylinder 2 to introduce nitrogen gas at a pressure of 0.6 MPa into hydrophilic column 7 and maintain it for 20 minutes; at the same time, use nano-chromatographic pump 5 to pump 6 μL of equilibration buffer through the trapping column 8 and the analytical column 9 in sequence to equilibrate the trapping column 8 and the analytical column 9.

[0080] S3.6 Switch the first ten-way valve 10 to the valve position so that the outlet of the loading pump 4 and the inlet of the hydrophilic column 7 are connected. Switch the second ten-way valve 11 to the valve position so that the outlet of the hydrophilic column 7 and the inlet of the collecting column 8 are connected.

[0081] S3.7 Using loading pump 4, continuously pump elution buffer at a volume of 3 times that of hydrophilic column 7 sequentially through hydrophilic column 7 and trapping column 8 to elute the glycopeptides retained on hydrophilic column 7 and transfer them to trapping column 8. The enrichment of intact glycopeptides in the next sample is completed. At the same time, use nano-chromatographic pump 5 to pump 900 nanoliters of equilibration buffer through analytical column 9 to make analytical column 9 equilibrate in equilibration buffer.

[0082] S3.8 Control the position of the second ten-way valve 11 so that the outlet of the collection column 8 and the inlet of the analysis column 9 are connected, while the position of the first ten-way valve 10 remains unchanged;

[0083] S3.9. Using loading pump 4, continuously pump 30 times the column volume of loading buffer into hydrophilic column 7 until it reaches equilibrium. At the same time, use nano-chromatographic pump 5 to pump microliters of equilibration buffer 6 through the trapping column 8 and the analytical column 9 in sequence until the trapping column 8 and the analytical column 9 reach equilibrium.

[0084] In this embodiment, the loading buffer contains acetonitrile as the organic solvent (80% by volume), trifluoroacetic acid as the organic acid (0.1% by volume), and water as the remaining component. The elution buffer contains acetonitrile as the organic solvent (0% by volume), trifluoroacetic acid as the organic acid (0.1% by volume), and water as the remaining component. The equilibration buffer contains acetonitrile as the organic solvent (2% by volume), formic acid as the organic acid (0.1% by volume), and water as the remaining component. The mobile phase solvent composition at its lowest organic phase volume percentage is: 1% organic phase (acetonitrile), 0.1% organic acid (formic acid), and water as the remaining component. The mobile phase solvent composition at its highest organic phase volume percentage is: 90% organic phase (acetonitrile), 0.1% organic acid (formic acid), and water as the remaining component.

[0085] The mass spectrometry total ion chromatography (i.e., mass spectrometry analysis data) of a single injection of mouse brain protein hydrolysate obtained by the method of this embodiment is shown in the figure below. Figure 3 As shown. Analysis Figure 3 It is known that, in this embodiment, 407 unique intact glycopeptides from 196 unique glycosylation modification sites of 172 unique glycosylated proteins were identified from mouse brain protein hydrolysates, including 60 unique glycan modifications.

[0086] Example 5: This example is basically the same as Example 4, except that in this example, the mass of the first sample in step S2.2 is 10 micrograms, and the mass of the next sample in step S3.2 is 10 micrograms.

[0087] The mass spectrometry total ion chromatography (i.e., mass spectrometry analysis data) of a single injection of mouse brain protein hydrolysate obtained by the method of this embodiment is shown in the figure below. Figure 4 As shown. Analysis Figure 4 It is known that, in this embodiment, 1423 unique intact glycopeptides from 804 unique glycosylation modification sites of 320 unique glycosylated proteins were identified from mouse brain protein hydrolysates, including 172 unique glycan modifications.

[0088] Example 6: This example is basically the same as Example 5, except that: in this example, the first sample in step S2.2 is human bronchoalveolar lavage fluid protease hydrolysate, and the next sample in step S3.2 is human bronchoalveolar lavage fluid protease hydrolysate.

[0089] The mass spectrometry total ion chromatogram (i.e., mass spectrometry analysis data) of a human bronchoalveolar lavage fluid protease hydrolysate obtained by the method of this embodiment is shown in the figure below. Figure 5 As shown. Analysis Figure 5It is known that, in this embodiment, 1724 unique intact glycopeptides from 252 unique glycosylation modification sites of 158 unique glycosylated proteins were identified from human bronchoalveolar lavage fluid protease hydrolysate, including 299 unique glycan modifications.

[0090] Example 7: This example is basically the same as Example 5, except that: in this example, the pressure of nitrogen gas introduced into the hydrophilic column 7 from the high-pressure gas cylinder 2 in step S2.5 is 1 MPa, and the pressure of nitrogen gas introduced into the hydrophilic column 7 from the high-pressure gas cylinder 2 in step S3.5 is 1 MPa.

[0091] The mass spectrometry total ion chromatography (i.e., mass spectrometry analysis data) of a single injection of mouse brain protein hydrolysate obtained by the method of this embodiment is shown in the figure below. Figure 6 As shown. Analysis Figure 6 It is known that, in this embodiment, 1415 unique intact glycopeptides from 505 unique glycosylation modification sites of 311 unique glycosylated proteins were identified from mouse brain protein hydrolysates, including 164 unique glycan modifications.

[0092] Example 8: This example is basically the same as Example 7, except that in this example, the first sample in step S2.2 is a protein hydrolysate of human liver cancer tissue, and the next sample in step S3.2 is a protein hydrolysate of human liver cancer tissue.

[0093] The mass spectrometry total ion chromatogram (i.e., mass spectrometry analysis data) of a single injection of human liver cancer tissue protease hydrolysate obtained by the method of this embodiment is shown in the figure below. Figure 7 As shown. Analysis Figure 7 It is known that, in this embodiment, 1699 unique intact glycopeptides from 484 unique glycosylation modification sites of 296 unique glycosylated proteins were identified from human liver cancer tissue hydrolysate, including 153 unique glycan modifications.

[0094] Example 9: This example is basically the same as Example 7, except that in this example, the first sample in step S2.2 is a protein hydrolysate of normal human liver tissue, and the next sample in step S3.2 is a protein hydrolysate of normal human liver tissue.

[0095] The mass spectrometry total ion chromatogram (i.e., mass spectrometry analysis data) of a single injection of human normal liver tissue protease hydrolysate obtained by the method of this embodiment is shown in the figure below. Figure 8 As shown. Analysis Figure 8 It is known that, in this embodiment, 1770 unique intact glycopeptides from 458 unique glycosylation modification sites of 266 unique glycosylated proteins were identified from human normal liver tissue protease hydrolysates, including 180 unique glycan modifications.

[0096] In summary, the high-throughput fully automated online enrichment and separation analysis method for intact glycopeptides of the present invention can achieve rapid, fully automated, online enrichment and separation analysis of intact glycopeptides. Moreover, the operation during the enrichment process is completely controllable, which can greatly reduce the problems of high sample loss rate, poor enrichment repeatability and low liquid chromatography resolution caused by manual operation. Furthermore, the time required for the enrichment and separation analysis steps of intact glycopeptides can be superimposed to significantly reduce the overall process time, which has broad application prospects.

Claims

1. A high-throughput, fully automated online enrichment and separation analysis method for intact glycopeptides, characterized in that... First, a fully automated online enrichment and separation analysis system capable of simultaneous enrichment and separation analysis via a ten-way valve is built. Then, this fully automated online enrichment and separation analysis system is used to enrich intact glycopeptides in the first sample. Next, the same fully automated online enrichment and separation analysis system is used to enrich intact glycopeptides in the next sample while simultaneously performing separation analysis of intact glycopeptides in the previous sample. This process is repeated until the separation analysis of intact glycopeptides in the last sample is completed.

2. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 1, characterized in that... The fully automated online enrichment and separation analysis system includes a column oven, two 10-port valves, a high-pressure gas cylinder, three waste liquid pipes, two waste liquid containers, nine plugs, a loading pump, a nanoliter chromatography pump, a mass spectrometry electrospray ionization source, a mass spectrometer, a hydrophilic interaction liquid chromatography column (i.e., a hydrophilic column), a reversed-phase chromatography trapping column (i.e., a trapping column), and a reversed-phase chromatography analytical column (i.e., an analytical column). The loading pump is a plunger pump, and the nanoliter chromatography pump is a binary plunger pump. Each 10-port valve has ten ports. The two 10-port valves are respectively installed on the column oven and are referred to as the first 10-port valve and the second 10-port valve. The nine plugs are referred to as the first plug to the ninth plug. The three waste liquid pipes are referred to as the first waste liquid pipe, the second waste liquid pipe, and the third waste liquid pipe. The two waste liquid containers are referred to as the first waste liquid container and the second waste liquid container. The outlet of the high-pressure gas cylinder, the first... The first plug, second plug, third plug, fourth plug, fifth plug, sixth plug, first waste liquid inlet, loading pump outlet, and hydrophilic column inlet are installed in clockwise order at the ten ports of the first ten-way valve; controlling the valve position of the first ten-way valve enables the loading pump outlet and the hydrophilic column inlet to communicate. The seventh plug, eighth plug, ninth plug, second waste liquid inlet, hydrophilic column outlet, trapping column inlet, nanoliter chromatography pump outlet, analytical column inlet, trapping column outlet, and third waste liquid inlet are installed in clockwise order at the ten ports of the second ten-way valve; controlling the valve position of the second ten-way valve enables the trapping column outlet and the analytical column inlet to communicate. The analytical column outlet is connected to the mass spectrometry electrospray ionization source, the mass spectrometry electrospray ionization source is connected to the mass spectrometer, the first waste liquid outlet is connected to the first waste liquid tank, and the second and third waste liquid outlets are respectively connected to the second waste liquid tank.

3. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 2, characterized in that... The hydrophilic column, trapping column, and analytical column were prepared as follows: Three capillaries, each open at one end and with a porous sieve plate at the other, were connected to a high-pressure chamber. A suspension of hydrophilic packing material in methanol was added to the high-pressure chamber connected to the first capillary and sealed. A suspension of reversed-phase chromatography packing material in methanol was added to the high-pressure chamber connected to the second capillary and sealed. A suspension of reversed-phase chromatography packing material in methanol was added to the high-pressure chamber connected to the third capillary and sealed. Each high-pressure chamber was then immersed in water, and a plunger pump was used to pump water into each chamber at a pressure of 8... Methanol at 00 bar was loaded using a high-pressure homogenization method. Hydrophilic packing material was filled into the first capillary with one open end and a porous sieve plate at the other end; reversed-phase chromatography packing material was filled into the second capillary with one open end and a porous sieve plate at the other end; and reversed-phase chromatography packing material was filled into the third capillary with one open end and a porous sieve plate at the other end. Each capillary with one open end and a porous sieve plate at the other end was then vibrated using an ultrasonic instrument to ensure uniform packing. At this point, the first capillary with one open end and a porous sieve plate at the other end filled with hydrophilic packing material formed a hydrophilic column; the second capillary with one open end and a porous sieve plate at the other end filled with reversed-phase chromatography packing material formed a trapping column; and the third capillary with one open end and a porous sieve plate at the other end filled with reversed-phase chromatography packing material formed an analytical column.

4. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 3, characterized in that... The capillary tube, open at one end and with a porous sieve plate at the other end, is prepared by the following steps: Step A1: Select a suitable capillary tube according to the quality of the sample to be processed. The capillary tube can be a quartz capillary tube or a stainless steel capillary tube, and cut the capillary tube to the required length for later use. Step A2: Mix formamide and potassium silicate solution at a volume ratio of 1:50 to 50:1, shake thoroughly until completely mixed, centrifuge and retain the supernatant; Step A3: Insert one end of the prepared capillary tube into the supernatant in a direction perpendicular to the supernatant until a portion of the capillary tube, which is 0.5 to 1.0 cm long, is completely immersed in the supernatant. At this time, the supernatant will be drawn into the capillary tube under the action of capillary, causing the liquid level inside the capillary tube to rise. Maintain this state until the liquid level of the supernatant inside and outside the capillary tube stabilizes and no longer changes. Step A4: Take out the soaked capillary tube vertically, keeping the capillary tube vertical, wipe off the supernatant adhering to the outer wall of the capillary tube, and then place the capillary tube vertically in an oven for heating, so that the supernatant is fully solidified at one end of the capillary tube to form a porous sieve plate. At this time, a capillary tube with one end open and the other end having a porous sieve plate is obtained.

5. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 3, characterized in that... The hydrophilic filler is any one or a mixture of two of the following: hydrophilic interaction fillers and zwitterionic hydrophilic interaction fillers.

6. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 3, characterized in that... The reverse chromatography packing material is any one or a mixture of at least two of the following: C4 packing material, C8 packing material, C10 packing material, C18 packing material, and C30 packing material.

7. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 3, characterized in that... The alkyl chain length of the reversed-phase chromatography packing material in the trapping column is less than or equal to the alkyl chain length of the reversed-phase chromatography packing material in the analytical column.

8. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 3, characterized in that... The specific process for enriching intact glycopeptides in the first sample using this fully automated online enrichment and separation analysis system is as follows: S2.1 Control the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column; control the position of the second ten-way valve to connect the outlet of the trapping column and the inlet of the analysis column. S2.2 Dissolve the preset dose of the first injection sample (i.e., protein hydrolysate) in the loading buffer to obtain the first injection sample solution. The concentration of the first injection sample in the first injection sample solution is 1 nanogram per microliter to 1 milligram per microliter. S2.3 First, use a loading pump to load the first sample solution onto the hydrophilic column at a flow rate of 1-500 μL per minute. Then, use the loading pump to deliver 1-500 times the column volume of loading buffer to flush the hydrophilic column, eluting the non-glycopeptides in the first sample solution onto the hydrophilic column. Next, use a nano-chromatographic pump to pump the mobile phase solvent with an organic phase ratio that increases linearly over time into the trapping and analytical columns until the organic phase ratio in the mobile phase solvent reaches the preset maximum value. Clean the trapping and analytical columns before enrichment and separation. S2.4 Switch the position of the first ten-way valve to connect the outlet of the high-pressure gas cylinder with the inlet of the hydrophilic column, while keeping the position of the second ten-way valve unchanged. S2.

5. Use a loading pump to deliver 1 μL to 10 mL of elution buffer to flush the loading pump. Use a high-pressure gas cylinder to introduce high-pressure gas at a pressure of 0.1-10 MPa into the hydrophilic column and maintain this pressure for 0.1-300 minutes. Simultaneously, use a nanoliter chromatography pump to pump 1 nanoliter to 100 μL of equilibration buffer sequentially through the trapping column and the analytical column to equilibrate them. S2.6 Switch the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column, and switch the position of the second ten-way valve to connect the outlet of the hydrophilic column and the inlet of the collection column. S2.7 Using a loading pump, continuously pump 1-200 times the column volume of elution buffer through the hydrophilic column and the trapping column sequentially to elute the glycopeptides retained on the hydrophilic column and transfer them to the trapping column. The enrichment of intact glycopeptides in the first sample is completed. At the same time, use a nano-chromatographic pump to pump 1 nanoliter-100 microliters of equilibration buffer through the analytical column to make the analytical column equilibrate in the equilibration buffer. S2.8 Control the position of the 20th valve so that the outlet of the collection column and the inlet of the analysis column are connected, while the position of the 10th valve remains unchanged; S2.

9. Use a loading pump to continuously pump 1-200 times the column volume of loading buffer to the hydrophilic column to bring it to equilibrium; at the same time, use a nano-chromatographic pump to pump 1 nanoliter to 100 microliters of equilibration buffer sequentially through the trapping column and the analytical column to bring them to equilibrium.

9. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 3, characterized in that... The specific process of using this fully automated online enrichment and separation analysis system to enrich intact glycopeptides in the next injection sample while simultaneously performing separation analysis of intact glycopeptides in the previous injection sample is as follows: S3.1 Control the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column; control the position of the second ten-way valve to connect the outlet of the trapping column and the inlet of the analysis column. S3.2 Dissolve the next dose of sample (i.e., protein hydrolysate) in loading buffer to obtain a solution with a protein hydrolysate concentration of 1 ng / µL to 1 mg / µL as the next dose sample solution. S3.

3. Use a loading pump to load the next injection sample solution onto the hydrophilic column at a flow rate of 1-500 μL / min. Then, use the loading pump to deliver 1-500 times the column volume of loading buffer to flush the hydrophilic column, eluting the non-glycopeptides from the previous injection sample solution onto the hydrophilic column. Simultaneously, a nano-chromatographic pump pumps a mobile phase solvent with an organic phase ratio that increases linearly or piecewise linearly over time into the trap column and the analytical column until the organic phase ratio in the mobile phase solvent reaches the preset maximum value. During this process, the intact glycopeptides from the previous injection sample stored on the trap column are separated by the analytical column and then enter the mass spectrometry electrospray ionization source. The mass spectrometry electrospray ionization source ionizes the intact glycopeptides from the previous injection sample and sends them to the mass spectrometer. The mass spectrometer outputs the mass spectrometry analysis data of the intact glycopeptides from the previous injection sample. S3.4 Switch the position of the first ten-way valve to connect the outlet of the high-pressure gas cylinder with the inlet of the hydrophilic column, while keeping the position of the second ten-way valve unchanged. S3.

5. Use a loading pump to deliver 1 μL to 10 mL of elution buffer to flush the loading pump and its connecting tubing; use a high-pressure gas cylinder to introduce high-pressure gas at a pressure of 0.1-10 MPa into the hydrophilic column and maintain this pressure for 0.1-300 minutes; simultaneously, use a nanoliter chromatography pump to pump 1 nanoliter to 100 μL of equilibration buffer sequentially through the trapping column and the analytical column to equilibrate them. S3.6 Switch the position of the first ten-way valve to connect the outlet of the loading pump and the inlet of the hydrophilic column, and switch the position of the second ten-way valve to connect the outlet of the hydrophilic column and the inlet of the collection column. S3.7 Using a loading pump, continuously pump 1-200 times the column volume of elution buffer through the hydrophilic column and the trapping column sequentially to elute the glycopeptides retained on the hydrophilic column and transfer them to the trapping column. The enrichment of intact glycopeptides in the next sample is completed. At the same time, use a nano-chromatographic pump to pump 1 nanoliter-100 microliters of equilibration buffer through the analytical column to make the analytical column equilibrate in the equilibration buffer. S3.8 Control the position of the 20th valve so that the outlet of the collection column and the inlet of the analysis column are connected, while the position of the 10th valve remains unchanged; S3.

9. Use a loading pump to continuously pump 1-200 times the column volume of loading buffer to the hydrophilic column to bring it to equilibrium. At the same time, use a nano-chromatographic pump to pump 1 nanoliter to 100 microliters of equilibration buffer sequentially through the trapping column and the analytical column to bring them to equilibrium.

10. The high-throughput fully automated online enrichment and separation method for intact glycopeptides according to claim 8 or 9, characterized in that... The loading buffer contains 50%-99% organic solvent, 0%-10% organic acid, and the remainder is water. The elution buffer contains 0%-15% organic solvent, 0%-10% organic acid, and the remainder is water. The equilibration buffer contains 0%-15% organic solvent, 0%-10% organic acid, and the remainder is water. The mobile phase solvent at its lowest organic phase volume percentage consists of 0.1%-15% organic phase, 0%-10% organic acid, and the remainder is water. The mobile phase solvent at its highest organic phase volume percentage consists of 30%-99% organic phase, 0%-10% organic acid, and the remainder is water.

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