IgA hinge region glycosylation test sample batch processing system

Abstract

The application discloses an IgA hinge region glycosylation detection sample batch processing system, and provides an IgA hinge region glycosylation detection sample batch processing system, method, equipment and computer readable storage medium. The system and method provided by the application greatly shorten sample pretreatment time, improve analysis consistency, and realize high-throughput and high-specificity IgA hinge region O-glycosylation proteome analysis.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to an IgA hinge region glycosylation detection sample batch processing system. BACKGROUND

[0002] Glycosylation is a common post-translational modification of proteins in the process of protein formation, and more than 50% of proteins in the human body have glycosylation modification, which has an important influence on the structure and function of proteins.

[0003] A large number of studies have found that abnormal glycosylation is often closely related to diseases, including cancer and congenital sugar chain defects. Abnormal protein with sugar chain structure formed in the glycosylation modification process has been determined as a marker of viral diseases, diabetes, autoimmune diseases, cancer, genetic diseases and the like, which has important significance in the occurrence, progression and metastasis of diseases, and also provides a new diagnostic method for disease diagnosis and research.

[0004] IgA nephropathy is the most common primary glomerular disease in the world and is the main cause of chronic renal failure. The pathological change of IgA nephropathy is the deposition of immunoglobulin mainly IgA1 in the mesangial region of the glomerulus. IgA1 is a highly glycosylated molecule, and the increase of the level of IgA1 with abnormal O-glycosylation in the circulation plays an important role in the pathogenesis of IgA nephropathy. Therefore, the detection of abnormal O-glycosylation of IgA1 hinge region is crucial for the diagnosis of IgA nephropathy.

[0005] Mass spectrometry (MS) is a commonly used method for studying glycosylated proteins, but the current detection method for the O-glycosylation level of IgA1 hinge region has a long sample processing time, low throughput, complex operation, large sample loss, non-parallel operation and unguaranteed repeatability, and therefore it is an urgent problem to be solved in the field to provide a sample processing method which is simple, fast, efficient and high-throughput. SUMMARY

[0006] In order to make up for the deficiencies of the prior art, the application provides an IgA hinge region glycosylation detection sample batch processing system and method.

[0007] To achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0008] The first aspect of the application provides an IgA hinge region glycosylation detection sample batch processing system, which comprises an extraction unit, a reducing alkylation enzyme cutting unit, a washing unit, an oscillation centrifugation unit, a reagent adding unit and a heating unit.

[0009] The extraction unit is used for extracting IgA protein, and the reducing alkylation enzyme cutting unit is used for cutting the IgA protein into peptide segments.

[0010] The extraction unit comprises a mixing subunit, and the mixing subunit comprises a filter plate on which an ultrafiltration membrane is arranged.

[0011] Further, the ultrafiltration membrane is an Omega ultrafiltration membrane.

[0012] Further, the Omega ultrafiltration membrane is a 100K Omega ultrafiltration membrane.

[0013] Further, the filter plate comprises a 384-well filter plate, a 96-well filter plate, a 48-well filter plate, or a 24-well filter plate.

[0014] Further, the filter plate is selected from a 96-well filter plate.

[0015] Further, the extraction unit is connected with a washing unit, an oscillation centrifugation unit, and a reagent adding unit, and the extraction unit comprises:

[0016] a mixing subunit for mixing the sample with IgA immunization affinity beads, and filtering the mixture through a filter plate to obtain a combination of IgA protein and IgA immunization affinity beads;

[0017] a washing subunit for washing the combination of IgA protein and IgA immunization affinity beads obtained in the mixing subunit to obtain a purified combination of IgA protein and IgA immunization affinity beads;

[0018] a separation subunit for separating the purified combination of IgA protein and IgA immunization affinity beads obtained in the washing subunit to obtain IgA protein.

[0019] Further, the mixing subunit comprises a mixing device, and in the mixing subunit, the sample and the IgA immunization affinity beads are mixed through the mixing device, and then treated by an oscillation device and a centrifugation device in the oscillation centrifugation unit, and filtered through the filter plate to obtain the combination of IgA protein and IgA immunization affinity beads.

[0020] Further, the oscillation device is set at a speed of 800-1200 rpm and a time of 0.5-1.5 hours.

[0021] Further, the oscillation device is set at a speed of 1000 rpm and a time of 1 hour.

[0022] Further, the centrifugation device is set at a speed of 2000-4000 rpm and a time of 1-5 minutes.

[0023] Further, the centrifugation device is set at a speed of 3000 rpm and a time of 3 minutes.

[0024] Further, the volume ratio of the sample to the IgA immunization affinity beads is 2:1.

[0025] Further, the sample is selected from the group consisting of blood plasma.

[0026] Further, the separation unit comprises a displacement device, in which, after the reagent adding device in the reagent adding unit adds glycine into the combination of the purified IgA protein and the IgA immunization affinity beads, the combination is treated by the oscillation device and the centrifugal device in the oscillation centrifugal unit, so as to obtain the IgA protein.

[0027] Further, the lower receiving plate is displaced by the displacement device before the glycine is added by the reagent adding device.

[0028] Further, the time of the oscillation device is set to 30-50 minutes.

[0029] Further, the time of the oscillation device is set to 40 minutes.

[0030] Further, the speed of the centrifugal device is set to 2000-4000g, and the time is set to 1-5 minutes.

[0031] Further, the speed of the centrifugal device is set to 3000g, and the time is set to 3 minutes.

[0032] Further, the glycine elution is repeated 4 times.

[0033] Further, in the washing subunit, the combination of the IgA protein and the IgA immunization affinity beads is washed by the washing device in the washing unit, and then treated by the centrifugal device in the oscillation centrifugal unit, so as to obtain the combination of the purified IgA protein and the IgA immunization affinity beads.

[0034] Further, the washing device in the washing unit comprises a PBS washing device and a mass spectrum water washing device, and the combination of the sample and the IgA immunization affinity beads is washed by the PBS washing device and the mass spectrum water washing device.

[0035] Further, the volume ratio of the PBS / mass spectrum water to the combination in the washing device is 1:1.

[0036] Further, the speed of the centrifugal device is set to 800-1200g, and the time is set to 1-3 minutes.

[0037] Further, the speed of the centrifugal device is set to 1000g, and the time is set to 1 minute.

[0038] Further, the combination of the sample and the IgA immunization affinity beads is washed by the washing device for 2-4 times.

[0039] Further, the combination of the sample and the IgA immunization affinity beads is washed by the washing device for 3 times.

[0040] Further, the extraction unit further comprises:

[0041] The affinity bead washing subunit is used for washing the IgA immunoadsorbent beads before the sample is mixed with the IgA immunoadsorbent beads, so as to obtain the washed IgA immunoadsorbent beads.

[0042] Further, in the affinity bead washing subunit, the IgA immunoadsorbent beads are washed by using the PBS washing device in the washing unit, and then treated by using the centrifugal device in the oscillation centrifugal unit.

[0043] Further, the volume ratio of the IgA immunoadsorbent beads to PBS is 1:10.

[0044] Further, the speed of the centrifugal device is set to 800-1200g, and the time is set to 1-3 minutes.

[0045] Further, the speed of the centrifugal device is set to 1000g, and the time is set to 1 minute.

[0046] Further, the IgA immunoadsorbent beads are washed by the PBS washing device for 3 times.

[0047] Further, the reducing alkylation enzyme cutting unit is connected with the washing unit, the oscillation centrifugal unit, the reagent adding unit and the heating unit, and the reducing alkylation enzyme cutting unit comprises:

[0048] The reducing unit is used for reducing the IgA protein extracted by the extraction unit, so as to obtain the reduced IgA protein.

[0049] The alkylation subunit is used for alkylating the reduced IgA protein, so as to obtain the alkylated IgA protein.

[0050] The enzyme cutting subunit is used for cutting the reduced and alkylated IgA protein, so as to obtain the enzyme-cut IgA protein.

[0051] The enzyme solution collecting subunit is used for collecting the enzyme solution.

[0052] Further, the reducing unit comprises a mixing device, and in the reducing unit, the IgA protein extracted by the extraction unit is dissolved in Tris, the DTT solution is added by using the sample adding device in the reagent adding unit, the treatment is performed by using the oscillation device and the centrifugal device in the oscillation centrifugal unit, and the treatment is performed by using the heating device in the heating unit, so as to obtain the reduced IgA protein.

[0053] Further, the concentration of the Tris is 10-30mM.

[0054] Further, the concentration of the Tris is 20mM.

[0055] Further, the concentration of the DTT solution is 1-3mol / L.

[0056] Further, the concentration of the DTT solution is 1mol / L.

[0057] Further, the heating device is set to 95-110℃, and the time is set to 5-15 minutes.

[0058] Further, the heating device is set to 100℃, and the time is set to 10 minutes.

[0059] Further, the alkylator unit comprises a reaction device, in the alkylator unit, the IAM solution is added to the reduced IgA protein through the sample adding device in the reagent adding unit, and the alkylated IgA protein is obtained through the oscillation device and the centrifugal device in the oscillation and centrifugal unit and reaction in the reaction device.

[0060] Further, the reaction device is set to light-proof reaction.

[0061] Further, the time of the reaction device is set to 30-60 minutes.

[0062] Further, the time of the reaction device is set to 45 minutes.

[0063] Further, the alkylator unit comprises a reaction device, in the alkylator unit, the IAM solution is added to the reduced IgA protein through the sample adding device in the reagent adding unit, and the alkylated IgA protein is obtained through the oscillation device and the centrifugal device in the oscillation and centrifugal unit and reaction in the reaction device.

[0064] Further, the mass ratio of the alkylated IgA protein to trypsin is 30-60:1.

[0065] Further, the mass ratio of the alkylated IgA protein to trypsin is 50:1.

[0066] Further, in the enzyme solution collecting unit, the IgA protein after enzyme digestion is treated by the centrifugal device in the oscillation and centrifugal unit to obtain the enzyme solution.

[0067] Further, the speed of the centrifugal device is set to 1000-5000g, and the time is set to 2-8 minutes.

[0068] Further, the speed of the centrifugal device is set to 3000g, and the time is set to 5 minutes.

[0069] Further, the reducing, alkylating and enzyme cutting unit further comprises:

[0070] The washing unit is used for washing the alkylated IgA protein obtained from the alkylator unit to obtain the washed alkylated IgA protein.

[0071] Further, in the washing subunit, the alkylated IgA protein is treated by the centrifugal device in the oscillation centrifugation subunit, and then Tris is added by the sample adding device in the reagent adding subunit, and then treated by the centrifugal device in the oscillation centrifugation subunit.

[0072] Further, the speed of the centrifugal device is set to 1000-5000g.

[0073] Further, the speed of the centrifugal device is set to 3000g.

[0074] Further, the IgA protein is IgA1 protein.

[0075] The second aspect of the present application provides an IgA hinge region glycosylation detection sample batch processing method, which comprises:

[0076] (1) extracting IgA protein;

[0077] (2) reducing alkylating enzyme cutting;

[0078] Step (1) comprises mixing the sample with IgA immunization affinity beads, centrifuging, and filtering the combination of IgA protein and IgA immunization affinity beads through a filter plate.

[0079] The filter plate is covered with an ultrafiltration membrane, and the filter plate is connected to a receiving plate.

[0080] Further, the ultrafiltration membrane is an Omega ultrafiltration membrane.

[0081] Further, the Omega ultrafiltration membrane is a 100K Omega ultrafiltration membrane.

[0082] Further, the filter plate comprises a 384-well filter plate, a 96-well filter plate, a 48-well filter plate, and a 24-well filter plate.

[0083] Further, the filter plate is selected from a 96-well filter plate.

[0084] Further, after mixing the sample with the IgA immunization affinity beads, the combination of IgA protein and IgA immunization affinity beads is obtained by further oscillation incubation and centrifugation.

[0085] Further, the oscillation speed is 800-1200rpm, and the time is 0.5-1.5 hours.

[0086] Further, the oscillation speed is 1000rpm, and the time is 1 hour.

[0087] Further, the centrifugal speed is 2000-4000rpm, and the time is 1-5 minutes.

[0088] Further, the centrifugal speed is 3000rpm, and the time is 3 minutes.

[0089] Further, the volume ratio of the sample to the IgA immunoaffinity beads is 2:1.

[0090] Further, the sample is selected from the group consisting of plasma.

[0091] Further, step (1) further comprises isolating the purified IgA protein from the mixture of the IgA protein and the IgA immunoaffinity beads.

[0092] Further, glycine is added to the mixture of the purified IgA protein and the IgA immunoaffinity beads, and the mixture is shaken and centrifuged to obtain the IgA protein.

[0093] Further, before the glycine is added, the lower receiving plate is replaced with a new one.

[0094] Further, the shaking time is 30-50 minutes.

[0095] Further, the shaking time is 40 minutes.

[0096] Further, the centrifugation speed is 2000-4000g, and the centrifugation time is 1-5 minutes.

[0097] Further, the centrifugation speed is 3000g, and the centrifugation time is 3 minutes.

[0098] Further, the glycine elution is repeated 4 times.

[0099] Further, step (1) further comprises washing the mixture of the IgA protein and the IgA immunoaffinity beads to obtain the mixture of the purified IgA protein and the IgA immunoaffinity beads.

[0100] Further, the mixture of the IgA protein and the IgA immunoaffinity beads is washed with PBS and / or mass spectrometry water, and centrifuged to obtain the mixture of the purified IgA protein and the IgA immunoaffinity beads.

[0101] Further, the mixture of the sample and the IgA immunoaffinity beads is washed with PBS and mass spectrometry water.

[0102] Further, the volume ratio of the PBS / mass spectrometry water to the mixture is 1:1.

[0103] Further, the centrifugation speed is 800-1200g, and the centrifugation time is 1-3 minutes.

[0104] Further, the centrifugation speed is 1000g, and the centrifugation time is 1 minute.

[0105] Further, the washing of the mixture of the sample and the IgA immunoaffinity beads is repeated 2-4 times.

[0106] Further, the washing of the mixture of the sample and the IgA immunoaffinity beads is repeated 3 times.

[0107] Further, step (1) further comprises washing the IgA immunoaffinity beads, centrifuging, to obtain washed IgA immunoaffinity beads before mixing the sample with the IgA immunoaffinity beads.

[0108] Further, the IgA immunoaffinity beads are washed with PBS.

[0109] Further, the volume ratio of the IgA immunoaffinity beads to PBS is 1:10.

[0110] Further, the centrifuging is performed at a speed of 800-1200g for 1-3 minutes.

[0111] Further, the centrifuging is performed at a speed of 1000g for 1 minute.

[0112] Further, the IgA immunoaffinity beads are washed for 3 times.

[0113] Further, step (2) comprises mixing the extracted IgA protein with a Tris, DTT solution, reducing the IgA protein.

[0114] Further, after mixing the extracted IgA protein with the Tris, DTT solution, the IgA protein is centrifuged and heated, and reduced.

[0115] Further, step (2) further comprises mixing the reduced IgA protein with an IAM solution, reacting, and alkylating the IgA protein.

[0116] Further, the reduced IgA protein is reacted with the IAM solution in the dark, and the IgA protein is alkylated.

[0117] Further, step (2) further comprises adding trypsin for enzymatic digestion.

[0118] Further, the mass ratio of the alkylated IgA protein to trypsin is 30-60:1.

[0119] Further, the mass ratio of the alkylated IgA protein to trypsin is 50:1.

[0120] Further, step (2) further comprises washing the alkylated IgA protein.

[0121] Further, the alkylated IgA protein is washed with Tris.

[0122] Further, the IgA protein is IgA1 protein.

[0123] The third aspect of the present application provides an IgA hinge region glycosylation detection sample batch processing device, comprising a memory and a processor.

[0124] The memory is configured to store program instructions;

[0125] The processor is configured to invoke the program instructions, when the program instructions are executed, for executing the IgA hinge region glycosylation detection sample batch processing method of the second aspect of the present application.

[0126] The fourth aspect of the present application provides a computer readable storage medium, which stores executable instructions, when the executable instructions are executed by a processor, the IgA hinge region glycosylation detection sample batch processing method of the second aspect of the present application is realized.

[0127] Advantages and beneficial effects of the present application:

[0128] The present application combines 96-well plates with IgA immunization affinity beads, controls solution residues in the enrichment process by centrifugation, reduces sample loss in the transfer process, shortens sample pretreatment time by whole plate processing, improves analysis consistency, and realizes high-throughput and high-specificity IgA1 hinge region O-glycosylation proteome analysis. BRIEF DESCRIPTION OF DRAWINGS

[0129] Figure 1 is the implementation process schematic diagram of the IgA hinge region glycosylation detection sample batch processing system;

[0130] Figure 2 is the IgA SDS-PAGE protein diagram extracted by the 96-well filter plate;

[0131] Figure 3 is the common sugar type primary mass spectrum diagram of the IgA1 hinge region processed by the method;

[0132] Figure 4 is the number of sugar numbers identified by the method and two other different sample pretreatment methods of the present application;

[0133] Figure 5 is the repeatability comparison diagram of the method and two other different sample pretreatment methods;

[0134] Figure 6 is the correlation diagram of the two within-group repeatability experiments of the method. DETAILED DESCRIPTION

[0135] The following provides definitions of some terms used in the specification. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0136] In some of the flowcharts described in the description and claims of the present application and in the above-mentioned figures, a plurality of operations are included which occur in a particular order, but it should be clearly understood that these operations can be performed in the order in which they appear herein or in parallel, and the serial numbers of the operations, such as 101, 102, etc., are merely used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these flowcharts can include more or fewer operations, and the operations can be performed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in the present application are used to distinguish different messages, devices, modules, etc., and do not represent the order of precedence, nor do "first" and "second" represent different types.

[0137] The technical solutions in the embodiments of the present application will be described clearly and completely in the following with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0138] Figure 1 is an implementation flowchart of the IgA hinge region glycosylation detection sample batch processing system, specifically, the system comprises: an extraction unit, a reducing alkylation enzyme cutting unit, a washing unit, an oscillation centrifugation unit, a reagent adding unit, and a heating unit:

[0139] 101: the extraction unit;

[0140] 102: the reducing alkylation enzyme cutting unit;

[0141] In an embodiment, the extraction unit is connected with the washing unit, the oscillation centrifugation unit, and the reagent adding unit, and the extraction unit is used to extract IgA protein, comprising:

[0142] The affinity bead washing subunit is used to wash the IgA immunoadsorbent beads before the sample is mixed with the IgA immunoadsorbent beads, to obtain washed IgA immunoadsorbent beads;

[0143] The mixing subunit is used to mix the sample with the IgA immunoadsorbent beads, and the combination of the IgA protein and the IgA immunoadsorbent beads is obtained by filtering through a filter plate;

[0144] The washing subunit is used to wash the combination of the IgA protein and the IgA immunoadsorbent beads obtained in the mixing subunit, to obtain the combination of the purified IgA protein and the IgA immunoadsorbent beads;

[0145] The separation subunit is used to separate the combination of the purified IgA protein and the IgA immunoadsorbent beads obtained in the washing subunit, to obtain the IgA protein.

[0146] The term "sample" as used herein refers to a composition obtained or derived from a subject (e.g., an individual of interest) that contains cells and / or other molecular entities to be characterized and / or identified according to, for example, physical, biochemical, chemical, and / or physiological characteristics. Samples include, but are not limited to, platelets, serum, plasma.

[0147] In a specific embodiment of the present application, the sample is selected from the group consisting of plasma.

[0148] In the affinity bead washing subunit, the IgA immunoaffinity beads are washed using the PBS washing device in the washing unit, and then treated using the centrifugation device in the oscillation centrifugation unit.

[0149] The volume ratio of IgA immunoaffinity beads to PBS is 1:10.

[0150] In an embodiment, the speed of the centrifugation device is set to 800-1200g, for example, it can be 800g, 900g, 1000g, 1100g, 1200g, and a range between any two of the above; the time is set to 1-3 minutes, for example, it can be 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, and a range between any two of the above.

[0151] In a specific embodiment of the present application, the speed of the centrifugation device is set to 1000g, and the time is set to 1 minute.

[0152] In an embodiment, the PBS washing device can wash the IgA immunoaffinity beads for 2 times, 3 times, 4 times, 5 times.

[0153] In a specific embodiment of the present application, the PBS washing device washes the IgA immunoaffinity beads for 3 times.

[0154] The mixing subunit comprises a mixing device. In the mixing subunit, after the sample and the IgA immunoaffinity beads are mixed by the mixing device, they are treated by the oscillation device and the centrifugation device in the oscillation centrifugation unit, and then filtered by the filter plate to obtain the combination of IgA protein and IgA immunoaffinity beads.

[0155] In an embodiment, the speed of the oscillation device is set to 800-1200rpm, for example, it can be 800rpm, 900rpm, 1000rpm, 1100rpm, 1200rpm, and a range between any two of the above; the time is set to 0.5-1.5 hours, for example, it can be 0.5 hours, 1 hour, 1.5 hours, and a range between any two of the above.

[0156] In a specific embodiment of the present application, the speed of the oscillation device is set to 1000rpm, and the time is set to 1 hour.

[0157] In one embodiment, the speed of the centrifugal device is set to 2000-4000 rpm, for example, it can be 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, and a range between any two of them; the time is set to 1-5 minutes, for example, it can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, and a range between any two of them.

[0158] In a specific embodiment of the application, the speed of the centrifugal device is set to 3000 rpm, and the time is set to 3 minutes.

[0159] In the washing subunit, the combination of IgA protein and IgA immunoaffinity beads is washed by the washing device in the washing unit, and then treated by the centrifugal device in the oscillation centrifugal unit to obtain the purified combination of IgA protein and IgA immunoaffinity beads.

[0160] In one embodiment, the speed of the centrifugal device is set to 800-1200 g, for example, it can be 800 g, 900 g, 1000 g, 1100 g, 1200 g, and a range between any two of them; the time is set to 1-3 minutes, for example, it can be 1 minute, 2 minutes, 3 minutes, and a range between any two of them.

[0161] In a specific embodiment of the application, the speed of the centrifugal device is set to 1000 g, and the time is set to 1 minute.

[0162] In one embodiment, the washing device washes the sample and IgA immunoaffinity bead mixture for 2-4 times, for example, it can be washed for 2 times, 3 times, 4 times.

[0163] In a specific embodiment of the application, the washing device washes the sample and IgA immunoaffinity bead mixture for 3 times.

[0164] The separation subunit comprises a displacement device. In the separation subunit, glycine is added to the purified combination of IgA protein and IgA immunoaffinity beads by the reagent adding device in the reagent adding unit, and then treated by the oscillation device and the centrifugal device in the oscillation centrifugal unit to obtain the IgA protein.

[0165] In one embodiment, the time of the oscillation device is set to 30-50 minutes, for example, it can be 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, and a range between any two of them.

[0166] In a specific embodiment of the application, the time of the oscillation device is set to 40 minutes.

[0167] In one embodiment, the centrifugal device is set at a speed of 2000-4000g, for example, it can be 2000g, 2500g, 3000g, 3500g, 4000g, and a range between any two of the above; the time is set to 1-5 minutes, for example, it can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, and a range between any two of the above.

[0168] In a specific embodiment of the present application, the centrifugal device is set at a speed of 3000g and a time of 3 minutes.

[0169] In one embodiment, the reducing alkylation enzyme cutting unit is connected with a cleaning unit, an oscillation centrifugal unit, a reagent adding unit, and a heating unit, and the reducing alkylation enzyme cutting unit comprises:

[0170] The reducing unit is used for reducing the IgA protein extracted by the extraction unit to obtain reduced IgA protein;

[0171] The alkylation unit is used for alkylating the reduced IgA protein to obtain alkylated IgA protein;

[0172] The enzyme cutting unit is used for cutting the reduced and alkylated IgA protein to obtain cut IgA protein;

[0173] The enzyme solution collecting unit is used for collecting the enzyme solution.

[0174] The reducing unit comprises a mixing device, and in the reducing unit, the IgA protein extracted by the extraction unit is dissolved in Tris, DTT solution is added by the sample adding device in the reagent adding unit, is treated by the oscillation device and the centrifugal device in the oscillation centrifugal unit, and is treated by the heating device in the heating unit to obtain reduced IgA protein.

[0175] In the present application, DTT is DL-Dithiothreitol, and its Chinese name is dithiothreitol. Its molecular formula is C4H 10 O2S2, and its molecular weight is 154.25.

[0176] In one embodiment, the concentration of the Tris is 10-30mM, for example, it can be 10mM, 15mM, 20mM, 25mM, 30mM, and a range between any two of the above.

[0177] In a specific embodiment of the present application, the concentration of the Tris is 20mM.

[0178] In one embodiment, the concentration of the DTT solution is 1-3mol / L, for example, it can be 1mol / L, 1.5mol / L, 2mol / L, 2.5mol / L, 3mol / L, and a range between any two of the above.

[0179] In a specific embodiment of the application, the concentration of the DTT solution is 1 mol / L.

[0180] In one embodiment, the heating device is set at a temperature of 95-110℃ and a time of 5-15 minutes, for example, the temperature can be 95℃, 98℃, 100℃, 102℃, 105℃, 107℃, 110℃, and a range between any two of the above; the time can be 5 minutes, 7 minutes, 10 minutes, 13 minutes, 15 minutes, and a range between any two of the above.

[0181] In a specific embodiment of the application, the heating device is set at a temperature of 100℃ and a time of 10 minutes.

[0182] The alkylator unit comprises a reaction device, in the alkylator unit, the IAM solution is added to the reduced IgA protein through the sample adding device in the reagent adding unit, treated by the oscillation device and the centrifugal device in the oscillation centrifugal unit, and reacted in the reaction device to obtain the alkylated IgA protein.

[0183] In one embodiment, the reaction device is set at a time of 30-60 minutes, for example, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, and a range between any two of the above.

[0184] In a specific embodiment of the application, the reaction device is set at a time of 45 minutes.

[0185] The enzyme cutting unit comprises an enzyme cutting device, in the enzyme cutting unit, the trypsin is added to the alkylated IgA protein through the sample adding device in the reagent adding unit, and the Tris is supplemented, and the enzyme cutting is assisted by the heating device in the heating unit to obtain the enzyme cut IgA protein.

[0186] In one embodiment, the mass ratio of the alkylated IgA protein to the trypsin is 30-60:1, for example, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, and a ratio range between any two of the above.

[0187] In a specific embodiment of the application, the mass ratio of the alkylated IgA protein to the trypsin is 50:1.

[0188] In one embodiment, the centrifugal device is set at a speed of 1000-5000g for a time of 2-8 minutes; the speed can be, for example, 1000g, 2000g, 3000g, 4000g, 5000g, and a range between any two of the above; the time can be, for example, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, and a range between any two of the above.

[0189] In a specific embodiment of the application, the centrifugal device is set at a speed of 3000g for a time of 5 minutes.

[0190] In one embodiment, the IgA protein comprises an IgA1 protein, an IgA2 protein.

[0191] In a specific embodiment of the application, the IgA protein is an IgA1 protein.

[0192] The application provides an IgA hinge region glycosylation detection sample batch processing device, comprising a memory and a processor;

[0193] The memory is used to store program instructions;

[0194] The processor is used to call the program instructions, and when the program instructions are executed, the IgA hinge region glycosylation detection sample batch processing method described above is executed.

[0195] In one embodiment, the device is a computer device, which can be a terminal, comprising a processor, a memory connected through a system bus; further comprising a network interface, a display screen and an input device. Wherein, the processor of the computer device is used to provide computing and control capabilities. The memory of the computer device comprises a non-volatile storage medium and an internal memory. The non-volatile storage medium stores an operating system and a computer program. The internal memory provides an environment for the operating system and the computer program in the non-volatile storage medium to run. The network interface of the computer device is used to communicate with external terminals through network connection. The display screen of the computer device can be a liquid crystal display screen or an electronic ink display screen. The input device of the computer device can be a touch layer covering the display screen, or a key, trackball or touchpad arranged on the shell of the computer device, or an external keyboard, touchpad or mouse, etc.

[0196] The above device is only part of the structure related to the scheme of the present application, and does not constitute a limitation on the computer device to which the scheme of the present application is applied. The specific computer device can include more or fewer components than those shown in the figure, or combine certain components, or have a different component arrangement.

[0197] The present invention provides a computer-readable storage medium having executable instructions stored thereon, which, when executed by a processor, implement the above-described batch processing method for detecting glycosylation in the IgA hinge region.

[0198] In one embodiment, the computer-readable storage medium may be a memory such as FRAM, ROM, PROM, EPROM, EEPROM, flash memory, magnetic surface memory, optical disk, or CD-ROM; or it may be a device that includes one or any combination of the above-mentioned memories.

[0199] In one implementation, the executable instructions may take the form of a program, software, software module, script, or code, written in any form of programming language (including compiled or interpreted languages, or declarative or procedural languages), and may be deployed in any form, including as a standalone program or as a module, component, subroutine, or other unit suitable for use in a computing environment.

[0200] As an example, executable instructions may, but do not necessarily, correspond to files in a file system. They may be stored as part of a file that holds other programs or data, for example, in one or more scripts in a Hyper Text Markup Language (HTML) document, in a single file dedicated to the program in question, or in multiple collaborating files (e.g., files that store one or more modules, subroutines, or code sections).

[0201] As an example, executable instructions can be deployed to execute on a single computing device, or on multiple computing devices located in one location, or on multiple computing devices distributed across multiple locations and interconnected via a communication network.

[0202] In this invention, the terms "an embodiment," "an example," "a specific embodiment," or "a particular embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. Furthermore, the described specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described herein, as well as the features of those different embodiments or examples.

[0203] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0204] Example

[0205] Extraction unit

[0206] Affinity Bead Washing Sub-unit: Add 10 μl of immunoaffinity beads to a 96-well 100K filter plate via the sample addition device, connect a 96-well receiving plate, add 100 μl of PBS via the reagent addition device in the reagent addition unit, wash with the PBS washing device in the washing unit, centrifuge at 1000g for 1 minute using the centrifuge device in the shaking centrifugation unit, discard the waste liquid, and repeat the washing device washing 3 times.

[0207] Mixed sample unit: Add 20 μl of plasma to a 96-well 100K filter plate via the sample addition device, add PBS to the total liquid volume of 200 μl via the reagent addition device in the reagent addition unit, shake at 1000 rpm for 1 h via the shaking device in the shaking centrifugation unit, centrifuge at 3000 rpm for 3 minutes, and discard the waste liquid.

[0208] Cleaning subunit: Add 200 μl PBS to the PBS cleaning device in the cleaning unit via the reagent addition device in the reagent addition unit, then centrifuge at 1000g for 1 minute via the centrifuge device in the shaking centrifugation unit, discard the waste liquid, and repeat 3 times;

[0209] 200 μl of mass spectrometer water was added to the mass spectrometer water cleaning device in the cleaning unit via the reagent addition device in the reagent addition unit. After cleaning, the mixture was centrifuged at 1000g for 1 minute via the centrifuge device in the shaking centrifugation unit. The waste liquid was discarded. This process was repeated 3 times.

[0210] Separation subunit: Replace the lower well plate with a new 96-well plate using the replacement device, add 50 μl of glycine using the reagent addition device in the reagent addition unit, shake for 40 min using the shaking device in the shaking centrifugation unit, centrifuge at 3000g for 3 minutes, and repeat 4 times.

[0211] Reductive alkylation enzyme digestion unit

[0212] Reduction subunit: After the protein extracted by the extraction unit is lyophilized, it is reconstituted in 200 μl of 20 mM Tris in the dissolution device, and 4 μl of 1 mol / L DTT solution is added through the sample loading device. After vortexing and centrifugation by the shaking and centrifugation devices in the shaking and centrifugation unit, it is heated at 100°C for 10 minutes in the heating device in the heating unit.

[0213] Alkylation subunit: After the protein reduced by the reduction subunit is cooled, 10 μl of 1 mol / L IAM solution is added through the sample loading device. After vortexing and centrifugation by the shaking device and centrifugation device in the shaking centrifugation unit, the protein is reacted in the reaction device in the dark for 45 minutes.

[0214] Enzyme digestion unit: Trypsin was added via a sample loading device at a mass ratio (sample:enzyme = 50:1), and Tris was added to a final volume of 30 μl via the sample loading device. The mixture was stirred to ensure that the liquid saturated the entire membrane. Enzyme digestion was performed twice for 1 min with the assistance of the heating device in the heating unit.

[0215] Enzyme hydrolysate collection subunit: The enzyme hydrolysate is obtained by centrifuging at 3000g for 5 minutes in the centrifugation unit until the membrane is clean and free of liquid.

[0216] Specific methods

[0217] (1) Add 10 μl of immunoaffinity beads to a 96-well 100K filter plate, attach a 96-well receiving plate, add 100 μl of PBS to each well using a pipette, wash, centrifuge at 1000g for 1 minute, discard the waste liquid, and repeat the washing 3 times.

[0218] (2) Add 20 μl of plasma to each well and add PBS to make the total liquid volume 200 μl, shake at 1000 rpm for 1 h;

[0219] (3) Centrifuge at 3000 rpm for 3 minutes and discard the waste liquid;

[0220] (4) Add 200 μl of PBS to each well of the pipette and wash, then centrifuge at 1000g for 1 minute, discard the waste liquid, and repeat 3 times;

[0221] (5) Add 200 μl of mass spectrometer water to each well of the array gun, wash, centrifuge at 1000g for 1 minute, discard the waste liquid, and repeat 3 times;

[0222] (6) Replace with a new 96-well plate, add 50 μl of glycine to each well, shake for 40 min, centrifuge at 3000 g for 3 min, and repeat four times.

[0223] (7) After the extracted protein was freeze-dried, it was reconstituted in 200 μl of 20 mM Tris, and 4 μl of 1 mol / L DTT solution was added. After vortexing and momentary separation, it was heated at 100 °C for 10 minutes.

[0224] (8) After cooling, add 10 μl of 1 mol / L IAM solution, vortex, briefly separate, and react in the dark for 45 min.

[0225] (9) After centrifugation at 3000g, add 200μl of 20mM Tris and repeatedly blow and beat for 30 times. Then, at 4℃ for 3000g for 10min, the tube is kept free of liquid. Repeat 3 times.

[0226] (10) Add trypsin according to the mass ratio (sample:enzyme = 50:1), add Tris to 30 μl, mix well, immerse the entire membrane in the liquid, and microwave on high for 1 min × 2 times.

[0227] (11) Centrifuge at 3000g for 5 minutes until the membrane is clean and free of liquid. The liquid after centrifugation is the enzymatic hydrolysate. After drying the peptides, reconstitute them in 10 μl of 0.1% FA solution. Take 2 μl of the solution and use a Thermo Orbitrap Explosher. TM The 480 instrument uses HCD fragmentation mode for mass spectrometry analysis.

[0228] Experimental results

[0229] Lanes 1-4 show the glycine elution buffer after four eluent washes from 10 μl plasma using a 100 kDa ultrafiltration plate; lanes 5-8 show the glycine elution buffer after four eluent washes from 20 μl plasma using a 100 kDa ultrafiltration plate. It can be seen that two glycine elutions remove most of the bound IgA protein. Combining the ultrafiltration plate with IgA immunoaffinity beads can enrich IgA protein in the plasma. Figure 2 ).

[0230] The first-order mass spectrum of common O-glucose forms in the IgA1 hinge region obtained by mass spectrometry analysis of plasma pools from IgA nephropathy patients is shown below. Figure 3 As shown.

[0231] Scheme A removed interference from the N-glycan chain using PNGase F after IgA protein extraction, and then enriched glycopeptides using HILIC material after reductive alkylation digestion; Scheme B involved reductive alkylation digestion of IgA protein followed by HILIC material enrichment of glycopeptides; Scheme C, the scheme used in this study, retained the N-glycan chain and did not perform HILIC enrichment. This sample pretreatment scheme can identify more types of glycopeptides. Figure 4 ).

[0232] The CV value of this sample pretreatment method is significantly lower than that of methods A and B, indicating that the reproducibility of this application method is strong. Figure 5 ).

[0233] The Pearson correlation between the two replicate experiments in this application is good, and its R0 2 =0.956 ( Figure 6 ).

[0234] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An IgA hinge region glycosylation detection sample batch processing system characterized by, The system comprises an extraction unit, a reducing alkylated enzyme cutting unit, a cleaning unit, an oscillation centrifugal unit, a reagent adding unit and a heating unit; The extraction unit is used for extracting IgA protein, and the reducing alkylated enzyme cutting unit is used for cutting the IgA protein into peptide segments; The extraction unit comprises a mixing unit, and the mixing unit comprises a filter plate on which an ultrafiltration membrane is arranged; The extraction unit is connected with the cleaning unit, the oscillation centrifugal unit and the reagent adding unit, and the extraction unit comprises: An affinity bead cleaning subunit is configured to clean IgA immunization affinity beads before the sample is mixed with the IgA immunization affinity beads, so as to obtain cleaned IgA immunization affinity beads; the IgA immunization affinity beads are cleaned by using a PBS cleaning device in the cleaning unit, and then treated by using a centrifugal device in the oscillation centrifugal unit; A mixing unit is configured to mix the sample with the IgA immunization affinity beads, and the mixture of the IgA protein and the IgA immunization affinity beads is obtained by filtering through a filter plate; the mixing unit comprises a mixing device; in the mixing unit, the sample and the IgA immunization affinity beads are mixed by using the mixing device, and then treated by using an oscillation device and a centrifugal device in the oscillation centrifugal unit, and finally filtered through the filter plate to obtain the mixture of the IgA protein and the IgA immunization affinity beads; A cleaning subunit is configured to clean the mixture of the IgA protein and the IgA immunization affinity beads obtained in the mixing unit, so as to obtain purified IgA protein and IgA immunization affinity beads; the mixture of the IgA protein and the IgA immunization affinity beads is cleaned by using a cleaning device in the cleaning unit, and then treated by using a centrifugal device in the oscillation centrifugal unit, so as to obtain the purified IgA protein and IgA immunization affinity beads; the cleaning device in the cleaning unit comprises a PBS cleaning device and a mass spectrometry water cleaning device; the mixture of the sample and the IgA immunization affinity beads is cleaned by using the PBS cleaning device and the mass spectrometry water cleaning device; A separation subunit is configured to separate the purified mixture of the IgA protein and the IgA immunization affinity beads obtained in the cleaning subunit, so as to obtain IgA protein; the separation subunit comprises a displacement device; in the separation subunit, glycine is added to the purified mixture of the IgA protein and the IgA immunization affinity beads by using a reagent adding device in the reagent adding unit, and then treated by using an oscillation device and a centrifugal device in the oscillation centrifugal unit, so as to obtain the IgA protein; The reducing alkylated enzyme cutting unit is connected with the cleaning unit, the oscillation centrifugal unit, the reagent adding unit and the heating unit, and the reducing alkylated enzyme cutting unit comprises: A reducing subunit is configured to reduce the IgA protein extracted by the extraction unit, so as to obtain reduced IgA protein; the reducing subunit comprises a mixing device; in the reducing subunit, the IgA protein extracted by the extraction unit is dissolved in Tris, DTT solution is added by using a sample adding device in the reagent adding unit, and then treated by using an oscillation device and a centrifugal device in the oscillation centrifugal unit, and finally treated by using a heating device in the heating unit, so as to obtain the reduced IgA protein. The alkylator unit is used for alkylating the reduced IgA protein to obtain an alkylated IgA protein. The alkylator unit comprises a reaction device. In the alkylator unit, the IAM solution is added to the reduced IgA protein through the sample adding device in the reagent adding unit, and is treated by the oscillation device and the centrifugal device in the oscillation centrifugal unit, and is reacted in the reaction device to obtain the alkylated IgA protein. The enzyme cutting unit is used for cutting the reduced alkylated IgA protein to obtain an enzyme cut IgA protein. The enzyme cutting unit comprises an enzyme cutting device. In the enzyme cutting unit, the trypsin is added to the alkylated IgA protein through the sample adding device in the reagent adding unit, and the Tris is supplemented, and the enzyme cutting is assisted by the heating device in the heating unit to obtain the enzyme cut IgA protein. The enzyme solution collecting unit is used for collecting the enzyme solution. The enzyme cut IgA protein is treated by the centrifugal device in the oscillation centrifugal unit to obtain the enzyme solution.

2. The system of claim 1, wherein, The ultrafiltration membrane is an Omega ultrafiltration membrane.

3. The system of claim 2, wherein, The Omega ultrafiltration membrane is a 100K Omega ultrafiltration membrane.

4. The system of claim 1, wherein, The filter plate comprises a 384-well filter plate, a 96-well filter plate, a 48-well filter plate, and a 24-well filter plate.

5. The system of claim 4, wherein, The filter plate is selected from a 96-well filter plate.

6. The system of claim 1, wherein, The speed of the oscillation device in the mixing unit is set to 800-1200 rpm, and the time is set to 0.5-1.5 hours.

7. The system of claim 6, wherein, The speed of the oscillation device in the mixing unit is set to 1000 rpm, and the time is set to 1 hour.

8. The system of claim 1, wherein, The speed of the centrifugal device in the mixing unit is set to 2000-4000 rpm, and the time is set to 1-5 minutes.

9. The system of claim 8, wherein, The speed of the centrifugal device in the mixing unit is set to 3000 rpm, and the time is set to 3 minutes.

10. The system of claim 1, wherein, The volume ratio of the sample to the IgA immunization affinity beads is 2:

1.

11. The system of claim 1, wherein, The sample is selected from plasma.

12. The system of claim 1, wherein, Before the glycine is added by the reagent adding device in the separation unit, the lower receiving plate is replaced by the replacement device.

13. The system of claim 1, wherein, The time of the oscillation device in the separation unit is set to 30-50 minutes.

14. The system of claim 13, wherein, The time of the oscillation device in the separation unit is set to 40 minutes.

15. The system of claim 1, wherein, The speed of the centrifugal device in the separation unit is set to 2000-4000 g, and the time is set to 1-5 minutes.

16. The system of claim 15, wherein, The speed of the centrifugal device in the separation unit is set to 3000 g, and the time is set to 3 minutes.

17. The system of claim 1, wherein, The glycine elution is repeated 4 times.

18. The system of claim 1, wherein, The volume ratio of PBS / mass spectrometry water to the binder in the washing device in the washing unit is 1:

1.

19. The system of claim 1, wherein, The speed of the centrifugal device in the washing unit is set to 800-1200 g, and the time is set to 1-3 minutes.

20. The system of claim 19, wherein, The speed of the centrifugal device in the washing unit is set to 1000 g, and the time is set to 1 minute.

21. The system of claim 1, wherein, The sample and IgA immunization affinity bead mixture is washed repeatedly 2-4 times in the washing device in the washing unit.

22. The system of claim 21, wherein, The sample and IgA immunization affinity bead mixture is washed repeatedly 3 times in the washing device in the washing unit.

23. The system of claim 1, wherein, The volume ratio of the IgA immunization affinity beads to PBS in the affinity bead washing unit is 1:

10.

24. The system of claim 1, wherein, The speed of the centrifugal device in the affinity bead washing unit is set to 800-1200 g, and the time is set to 1-3 minutes.

25. The system of claim 24, wherein, The speed of the centrifugal device in the affinity bead washing unit is set to 1000 g, and the time is set to 1 minute.

26. The system of claim 1, wherein, The PBS cleaning device in the affinity bead cleaning subunit cleans the IgA immune affinity beads for 3 times.

27. The system of claim 1, wherein, The concentration of the Tris in the reduction subunit is 10-30 mM.

28. The system of claim 27, wherein, The concentration of the Tris in the reduction subunit is 20 mM.

29. The system of claim 1, wherein, The concentration of the DTT solution in the reduction subunit is 1-3 mol / L.

30. The system of claim 29, wherein, The concentration of the DTT solution in the reduction subunit is 1 mol / L.

31. The system of claim 1, wherein, The temperature of the heating device in the reduction subunit is set to 95-110℃, and the time is set to 5-15 minutes.

32. The system of claim 31, wherein, The temperature of the heating device in the reduction subunit is set to 100℃, and the time is set to 10 minutes.

33. The system of claim 1, wherein, The reaction device in the alkylation subunit is set to avoid light reaction.

34. The system of claim 1, wherein, The time of the reaction device in the alkylation subunit is set to 30-60 minutes.

35. The system of claim 34, wherein, The time of the reaction device in the alkylation subunit is set to 45 minutes.

36. The system of claim 1, wherein, The mass ratio of the alkylated IgA protein to trypsin in the enzyme cutting subunit is 30-60:

1.

37. The system of claim 36, wherein, The mass ratio of the alkylated IgA protein to trypsin in the enzyme cutting subunit is 50:

1.

38. The system of claim 1, wherein, The speed of the centrifugal device in the enzyme solution collection subunit is set to 1000-5000 g, and the time is set to 2-8 minutes.

39. The system of claim 38, wherein, The speed of the centrifugal device in the enzyme solution collection subunit is set to 3000 g, and the time is set to 5 minutes.

40. The system of claim 1, wherein, The speed of the centrifugal device in the reduction, alkylation and enzyme cutting cleaning subunit is set to 1000-5000 g.

41. The system of claim 40, wherein, The speed of the centrifugal device in the reduction, alkylation and enzyme cutting cleaning subunit is set to 3000 g.

42. The system of claim 1, wherein, The IgA protein is IgA1 protein.

43. A batch processing method for IgA hinge region glycosylation detection samples, characterized in that, The method comprises: (1) extracting IgA protein; (2) reduction, alkylation and enzyme cutting; Step (1) comprises mixing the sample with IgA immune affinity beads, centrifuging, and filtering the combination of IgA protein and IgA immune affinity beads through a filter plate. An ultrafiltration membrane is coated on the filter plate, and the filter plate is connected to a receiving plate. Step (1) further comprises oscillation incubation after mixing the sample with IgA immune affinity beads, centrifuging, and obtaining the combination of IgA protein and IgA immune affinity beads. Step (1) further comprises separating and purifying the combination of IgA protein and IgA immune affinity beads to obtain IgA protein; adding glycine to the purified combination of IgA protein and IgA immune affinity beads, oscillating, and centrifuging to obtain IgA protein. Step (1) further comprises cleaning the combination of IgA protein and IgA immune affinity beads to obtain the purified combination of IgA protein and IgA immune affinity beads; using PBS and / or mass spectrometry water to clean the combination of IgA protein and IgA immune affinity beads, centrifuging to obtain the purified combination of IgA protein and IgA immune affinity beads; using PBS and mass spectrometry water to clean the combination of sample and IgA immune affinity beads; Step (1) further comprises cleaning the IgA immune affinity beads before mixing the sample with the IgA immune affinity beads, centrifuging to obtain the cleaned IgA immune affinity beads; and using PBS to clean the IgA immune affinity beads. Step (2) further comprises mixing the reduced IgA protein with IAM solution, reacting, and alkylating the IgA protein; the reduced IgA protein is reacted with IAM solution in the dark to alkylate the IgA protein. Step (2) further comprises mixing the reduced IgA protein with IAM solution, reacting, and alkylating the IgA protein; the reduced IgA protein is reacted with IAM solution in the dark to alkylate the IgA protein. Step (2) further comprises adding trypsin for enzymatic digestion. Step (2) further comprises washing the alkylated IgA protein; the alkylated IgA protein is washed with Tris.

44. The method of claim 43, wherein, The ultrafiltration membrane is an Omega ultrafiltration membrane.

45. The method of claim 44, wherein, The Omega ultrafiltration membrane is a 100K Omega ultrafiltration membrane.

46. The method of claim 43, wherein, The filter plate is selected from the group consisting of a 384-well filter plate, a 96-well filter plate, a 48-well filter plate, and a 24-well filter plate.

47. The method of claim 46, wherein, The filter plate is selected from the group consisting of a 96-well filter plate.

48. The method of claim 43, wherein, The speed of oscillation of the sample mixed with the IgA immunoaffinity beads in step (1) is 800-1200 rpm for 0.5-1.5 hours.

49. The method of claim 48, wherein, The speed of oscillation of the sample mixed with the IgA immunoaffinity beads in step (1) is 1000 rpm for 1 hour.

50. The method of claim 43, wherein, The speed of centrifugation of the sample mixed with the IgA immunoaffinity beads after incubation in step (1) is 2000-4000 rpm for 1-5 minutes.

51. The method of claim 50, wherein, The speed of centrifugation of the sample mixed with the IgA immunoaffinity beads after incubation in step (1) is 3000 rpm for 3 minutes.

52. The method of claim 43, wherein, The volume ratio of the sample to the IgA immunoaffinity beads is 2:

1.

53. The method of claim 43, wherein, The sample is selected from the group consisting of plasma.

54. The method of claim 43, wherein, The lower receiving plate is replaced with a new one before the addition of glycine in step (1).

55. The method of claim 43, wherein, The time of oscillation after the addition of glycine in step (1) is 30-50 minutes.

56. The method of claim 55, wherein, The time of oscillation after the addition of glycine in step (1) is 40 minutes.

57. The method of claim 43, wherein, The speed of centrifugation after the addition of glycine in step (1) is 2000-4000 g for 1-5 minutes.

58. The method of claim 57, wherein, The speed of centrifugation after the addition of glycine in step (1) is 3000 g for 3 minutes.

59. The method of claim 43, wherein, The glycine elution is repeated 4 times.

60. The method of claim 43, wherein, The volume ratio of PBS / mass spectrometry water to the combination used for washing the IgA protein and IgA immunoaffinity bead mixture in step (1) is 1:

1.

61. The method of claim 43, wherein, The speed of centrifugation of the IgA protein and IgA immunoaffinity bead mixture after washing in step (1) is 800-1200 g for 1-3 minutes.

62. The method of claim 61, wherein, The speed of centrifugation of the IgA protein and IgA immunoaffinity bead mixture after washing in step (1) is 1000 g for 1 minute.

63. The method of claim 43, wherein, The IgA protein and IgA immunoaffinity bead mixture is washed 2-4 times in step (1).

64. The method of claim 63, wherein, The IgA protein and IgA immunoaffinity bead mixture is washed 3 times in step (1).

65. The method of claim 43, wherein, The volume ratio of the IgA immunoaffinity beads to PBS in step (1) is 1:

10.

66. The method of claim 43, wherein, The speed of centrifugation of the IgA protein and IgA immunoaffinity bead mixture before washing in step (1) is 800-1200 g for 1-3 minutes.

67. The method of claim 66, wherein, The speed of centrifugation of the IgA protein and IgA immunoaffinity bead mixture before washing in step (1) is 1000 g for 1 minute.

68. The method of claim 67, wherein, The IgA immunoaffinity beads are washed 3 times.

69. The method of claim 43, wherein, Step (2) the mass ratio of the alkylated IgA protein to trypsin is 30-60:

1.

70. The method of claim 69, wherein, The mass ratio of the alkylated IgA protein to trypsin is 50:

1.

71. The method of claim 43, wherein, The IgA protein is an IgA1 protein.

72. An IgA hinge region glycosylation detection sample batch processing apparatus, comprising: The device comprises a memory and a processor; The memory is configured to store program instructions; The processor is configured to invoke the program instructions, which when executed, perform the IgA hinge region glycosylation detection sample batch processing method of any one of claims 43-71.

73. A computer-readable storage medium having stored thereon executable instructions that cause a processor-based device to: The executable instructions, when executed by the processor, implement the IgA hinge region glycosylation detection sample batch processing method of any one of claims 43-71.

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