High-flux in-gel enzymolysis peptide fragment enrichment kit and automatic enrichment method

Abstract

The invention discloses a high-flux in-gel enzymolysis peptide fragment enrichment kit and an automatic enrichment method, and belongs to the technical field of peptide fragment extraction, and the high-flux in-gel enzymolysis peptide fragment enrichment method adopts a high-flux tissue grinder and an automatic pipetting workstation; the high-flux in-gel enzymolysis peptide fragment enrichment method comprises the following steps: grinding a gel strip sample to obtain a colloidal particle sample, and preparing an in-station reagent of an automatic pipetting workstation; a reagent is automatically added through an automatic pipetting workstation, the colloidal particle sample is sequentially subjected to decoloration, cleaning, curing, reduction, closing, enzymolysis and elution, and a peptide fragment sample is obtained; salt ions and impurities in the peptide fragment sample are removed, and the peptide fragment sample is placed in a vacuum concentrator to be drained for standby application; on the basis of an automatic pipetting workstation and a 96-well plate, automatic in-gel enzyme digestion peptide fragment collection of large-scale samples is realized, the sample treatment time is greatly shortened, and the batch effect between the samples is greatly reduced; the automatic desalting step is added, so that the risk of chromatographic column blockage caused by colloidal particle residues in later detection is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of peptide extraction, more particularly to a high-throughput in-gel enzymatic digestion peptide enrichment kit and an automatic enrichment method. BACKGROUND

[0002] In-gel enzymatic digestion is to degrade the proteins remaining in the gel after electrophoresis into peptides under the action of various proteases. As a classic technology connecting gel electrophoresis and mass spectrometry identification, in-gel enzymatic digestion has long played an important role in proteomics research. However, the peptides obtained by enzymatic digestion are still preserved in the gel block, and the in-gel peptides still need to be extracted and processed.

[0003] The traditional peptide extraction manual operation has complicated steps and is highly dependent on the experience of the operator, and has inherent limitations such as low throughput, poor repeatability and low efficiency, which seriously restricts the application of in-gel enzymatic digestion technology in large-scale proteomics research. SUMMARY

[0004] The purpose of the present application is to solve the technical problems of low throughput and poor repeatability of the traditional peptide extraction method, and to provide an automatic high-throughput in-gel enzymatic digestion peptide enrichment kit and method.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme: The first aspect of the present application provides an automatic high-throughput in-gel enzymatic digestion peptide enrichment method, which adopts a high-throughput tissue grinder and an automatic pipetting workstation. The high-throughput in-gel enzymatic digestion peptide enrichment method comprises the following steps: Grinding the gel strip sample to obtain a gel particle sample, and configuring reagents in the automatic pipetting workstation; Adding reagents automatically through the automatic pipetting workstation, and sequentially performing decolorization, cleaning, solidification, reduction, blocking, enzymatic digestion and elution on the gel particle sample to obtain a peptide sample; Removing salt ions and impurities from the peptide sample, and placing it in a vacuum concentrator for drying.

[0006] Further, the automatic pipetting workstation is equipped with an operation table, a mechanical arm provided with a clamping jaw, and two 8-channel pipetting guns. The operation table of the automatic pipetting workstation contains a garbage can, a carrier containing a desalting column, a carrier containing 96-hole gun heads, a positive pressure module, two heating and shaking modules, a 96-hole plate containing a PVDF membrane column, two 1.2ml deep hole plates, a four-channel groove, and a plurality of plate positions. Among them, the 96-hole plate containing the PVDF membrane column is marked as the PDVF plate; the two 1.2ml deep hole plates are marked as the Waste plate and the Collection plate respectively; and the four-channel groove is marked as the waste liquid groove. The reagents include: a decolorizing solution, a cleaning solution, a solidifying solution, a reducing reagent, an alkylating reagent, an enzymatic solution, a termination solution, an eluent 1, an eluent 2, a cleaning solution D, a cleaning solution E, and an eluent F.

[0007] Further, the rubber strip sample is ground to obtain a rubber particle sample, and the specific operation steps are as follows: The rubber strip sample is transferred to a 2-ml grinding tube, grinding beads and a decolorizing solution are added, the grinding tube is placed in a high-throughput tissue grinder, and after grinding, the grinding beads are taken out to obtain a rubber particle sample; The rubber particle sample is placed in a PDVF plate and placed in a plate position containing a waste liquid tank.

[0008] Further, the rubber particle sample is decolorized and cleaned, and the specific operation steps are as follows: When decolorizing, the decolorizing solution is added, and the shaking and positive pressure treatment are repeated 3 times; When cleaning, the cleaning solution is added, and the shaking and positive pressure treatment are repeated 2 times.

[0009] Further, the rubber particle sample is solidified, and the specific operation steps are as follows: After adding the solidifying solution, shake at 500 rpm for 5 min, 0.06 MPa positive pressure for 30 s, and repeat 2 times.

[0010] Further, the rubber particle sample is reduced and blocked, and the specific operation steps are as follows: When the disulfide bond of the rubber particle sample is broken, the reducing reagent is added, and the shaking is performed at 1000 rpm for 1 h and the positive pressure is 0.06 MPa for 30 s; When the mercapto group of the rubber particle sample is blocked, the alkylating reagent is added, and the shaking is performed at 500 rpm for 45 min and the positive pressure is 0.06 MPa for 30 s.

[0011] Further, before enzymolysis, the rubber particle sample is subjected to secondary decolorization and secondary solidification, and the specific operation steps are as follows: After adding the cleaning solution, shake at 800 rpm for 5 min, 0.06 MPa positive pressure for 30 s, and repeat 2 times; When the secondary decolorization is performed, the decolorizing solution is added, and the shaking is performed at 800 rpm for 10 min and the positive pressure is 0.06 MPa for 30 s, and the process is repeated 3 times; When the secondary solidification is performed, the solidifying solution is added, and the shaking is performed at 500 rpm for 5 min and the positive pressure is 0.06 MPa for 30 s, and the process is repeated 2 times.

[0012] Further, the rubber particle sample is subjected to enzymolysis, and the specific operation steps are as follows: After adding the enzymatic solution, shake at 500 rpm for 1-2 h; After adding the termination solution, shake at 500 rpm for 30 s and 0.06 MPa positive pressure for 30 s.

[0013] Further, the colloidal particle sample is eluted, and the specific operation steps are as follows: After adding eluent 1, oscillate at 1000 rpm for 10 min, and apply 0.06 MPa positive pressure for 30 s; After adding eluent 2, oscillate at 1000 rpm for 10 min, and apply 0.06 MPa positive pressure for 30 s; The salt ions and impurities in the peptide segment sample are removed, and the specific operation steps are as follows: After the peptide segment sample is redissolved in 0.1% FA, a desalting column is added, and 0.06 MPa positive pressure is applied for 30 s; After adding cleaning solution D, 0.06 MPa positive pressure is applied for 30 s, and the operation is repeated twice; After adding cleaning solution E, 0.06 MPa positive pressure is applied for 30 s, and the operation is repeated twice; After adding eluent F, 0.06 MPa positive pressure is applied for 30 s, and the operation is repeated twice.

[0014] Another aspect of the present application provides an automatic high-throughput in-gel enzymatic peptide segment enrichment kit, which comprises: A decolorizing solution, preferably 50% acetonitrile and 50 mM ammonium bicarbonate; A cleaning solution, preferably 50 mM ammonium bicarbonate; A solidifying solution, preferably 100% acetonitrile; A reducing reagent, which is dithiothreitol or trichloroethyl phosphate, preferably 5-20 mM trichloroethyl phosphate; An alkylating reagent, preferably 10-55 mM chloroacetamide; An enzymatic solution, which is trypsin or a mixture of trypsin and recombinant lysine enzyme, preferably a mixture of trypsin and recombinant lysine enzyme; A termination solution, preferably 10 v / v% trifluoroacetic acid; A desalting column, the filler of which is a reversed-phase chromatographic filler, preferably C18; Eluent 1, preferably 60% acetonitrile and 0.1% trifluoroacetic acid; Eluent 2, preferably 90% acetonitrile and 0.1% trifluoroacetic acid; Cleaning solution D, preferably 100 v / v% ethyl acetate and 0.2 v / v% trifluoroacetic acid; Cleaning solution E, preferably 0.2 v / v% trifluoroacetic acid; Eluent F, preferably 5 v / v% ammonium hydroxide and 80 v / v% acetonitrile.

[0015] The beneficial effects of the present application are: the present application realizes large-scale sample automatic in-gel digestion peptide collection based on an automated pipetting workstation and a 96-well plate, increases the processing throughput to at most 96 samples at a time, greatly reduces the sample processing time and batch effects between samples; on the other hand, by adding an automated desalting step, the risk of chromatographic column blockage caused by gel particle residues in later detection is reduced; finally, through the optimized pretreatment scheme, the experimental period is significantly shortened, and the in-gel digestion process of at least 96 samples can be completed within 5 hours, which is nearly 10 times shorter than the prior art which takes at least 48 hours. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a schematic diagram of the operation platform plate position in the automated pipetting workstation in the embodiment of the present application; Figure 2 Figure 2 is a comparison chart of the effect of the gel particle sample before (left) and after (right) decolorization in the embodiment of the present application; Figure 3 Figure 3 is a mass spectrometer detection result total ion flow chromatogram (TIC) in the embodiment of the present application; Figure 4 Figure 4 is a gel strip sample protein identification result in the present application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than 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.

[0018] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present application are only used to explain the relative positional relationship, movement condition, etc. between components in a certain posture (as shown in the drawings), and if the certain posture changes, the directional indications also change accordingly.

[0019] In addition, if the description of "first", "second" and the like is involved in the embodiments of the present application, the description of "first", "second" and the like is only for the purpose of description, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second" can be explicitly or implicitly included at least one of the features. In addition, the meaning of "and / or" appearing throughout the text includes three parallel schemes, for example, "A and / or B" includes A scheme, or B scheme, or A and B scheme. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the realization of the ordinary skilled in the art, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor within the scope of protection required by the present application.

[0020] The test method used in the embodiments is a conventional method unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0021] Please refer to Figures 1 to 4 The embodiment of the present application shown provides an automatic high-throughput in-gel enzymatic peptide enrichment method, which aims to realize automatic in-gel enzymatic peptide collection of large-scale samples, eliminate the inconsistency of human operation through mechanical control, and promote the transformation of proteomics from "small-scale exploration" to "large-scale, high-precision discovery"; the automatic high-throughput in-gel enzymatic peptide enrichment method adopts a high-throughput tissue grinder and an automatic pipetting workstation; in the embodiment, the automatic pipetting workstation is at least equipped with: an operation table, a mechanical arm provided with a clamping jaw, two 8-channel pipetting guns; the operation table contains: a garbage can, a carrier loaded with a desalting column, a carrier loaded with 96-hole gun heads, a positive pressure module, two heating and shaking modules, a 96-hole plate containing a PVDF membrane column, two 1.2ml deep hole plates, a four-channel groove and a plurality of plate positions; the heating and shaking module is preferably controlled in the temperature range of 37-95℃, with a rotation speed of 0-1000rpm, and has the function of automatic temperature control; the 96-hole plate containing the PVDF membrane column is marked as the PDVF plate; the two 1.2ml deep hole plates are respectively marked as the Waste plate and the Collection plate; the four-channel groove is marked as the waste liquid groove; the above-mentioned PDVF plate, Collection plate and waste liquid groove are respectively located on the corresponding plate positions of the operation table, and the Waste plate is located on the heating and shaking module.

[0022] The automatic high-throughput in-gel enzymatic peptide enrichment method comprises the following steps: The gel strip sample is ground to obtain a gel particle sample, and the reagents in the automatic pipetting workstation are configured; In this step, the reagents include: decolorizing solution, cleaning solution, solidifying solution, reducing reagent, alkylating reagent, enzymatic hydrolysis solution, termination solution, eluent 1, eluent 2, cleaning solution D, cleaning solution E and eluent F.

[0023] During grinding, the rubber strip sample is first taken out of the -80℃ freezer; Transfer the adhesive strip sample to a 2ml grinding tube, add one grinding bead to each tube, preferably a 5mm 440C stainless steel bead; add decolorizing solution, preferably 300μl per tube; The grinding tube was placed in a high-throughput tissue homogenizer and ground at 30 Hz for 20 seconds, then allowed to stand for 20 seconds. This process was repeated twice. After grinding, the tube was centrifuged at high speed for 1 minute to remove the fragmented gel from the cap and tube wall to the bottom of the tube. 700 μL of decolorizing solution was added, and the steel beads were attracted out with a magnet to obtain an extremely fine gel sample. The ground gel sample is transferred to the wells of the PVDF plate, preferably with a peptide mass of 1µg-50μg per well; the PVDF plate is then moved to the plate position where the waste liquid tank is located in the automated pipetting workstation; it should be noted that in this embodiment, when the PVDF plate is moved to the plate position where the waste liquid tank is located, the two are combined to form a double-layer plate position.

[0024] The reagents are automatically added using an automated pipetting workstation, and the colloidal sample is sequentially decolorized, washed, solidified, reduced, blocked, enzymatically digested, and eluted to obtain peptide samples.

[0025] The purpose of decolorization and cleaning is to remove dyes and impurities, making the colloidal sample clean. During decolorization, the robotic arm controls the grippers to move the double-layer plate to the corresponding position of the positive pressure module and apply positive pressure, preferably 0.06 MPa for 30 seconds. The colloidal sample is then cleaned, and the double-layer plate is moved back. The automated pipetting workstation automatically pipes the solution, adding decolorizing solution to each well. The robotic arm controls the grippers to move the PVDF plate to the Waste plate of the heating and shaking module, preferably shaking at 25°C and 800 rpm for 10 minutes. The robotic arm controls the grippers to move the PVDF plate back to the plate position of the waste liquid tank. The robotic arm controls the grippers to move the double-layer plate to the corresponding position of the positive pressure module, preferably applying 0.06 MPa for 30 seconds, and then moves the double-layer plate back. This "double-layer plate relocation" means moving the PVDF plate and the waste liquid tank back to their corresponding positions on the operating table. The above steps are repeated 3 times to decolorize the colloidal sample.

[0026] During cleaning, the automated pipetting workstation automatically pipes the cleaning solution into each well; the robotic arm moves the gripper to the Waste plate on the heated and vibrating module of the automated pipetting workstation, preferably vibrating at 25°C and 800 rpm for 5 minutes; the robotic arm moves the gripper back to the waste liquid tank; the robotic arm moves the gripper to the corresponding position of the double-layer plate, preferably applying a positive pressure of 0.06 MPa for 30 seconds, and then moves the double-layer plate back; the above steps are repeated twice to clean the colloidal sample.

[0027] The purpose of curing is to further stabilize the sample structure and prevent protein loss. During curing, the automated pipetting workstation automatically pipes the curing solution into each well, preferably 500 μl per well. The robotic arm controls the grippers to move the PVDF plate to the Waste plate of the heated and oscillating module of the automated pipetting workstation, preferably oscillating at 25°C and 500 rpm for 5 min. The robotic arm controls the grippers to move the PVDF plate back to the plate position of the waste liquid tank. The robotic arm controls the grippers to move the double-layer plate to the corresponding position of the positive pressure module, preferably applying a positive pressure of 0.06 MPa for 30 s, and then moving the double-layer plate back. The above steps are repeated twice to cure the colloidal sample.

[0028] The purpose of reduction and blocking is to break disulfide bonds and block thiol groups, creating conditions for subsequent enzymatic digestion of colloidal samples. During reduction, the robotic arm controls the gripper to move the PVDF plate to the Waste plate in the heating and shaking module. The automated pipetting workstation automatically pipes the solution, adding the reducing reagent to each well, preferably 150 μl per well. The solution is heated and shaken at 37°C and 1000 rpm for 1 hour. The robotic arm controls the gripper to move the PVDF plate back to the plate position in the waste liquid tank. The robotic arm controls the gripper to move the double-layer plate to the corresponding position in the positive pressure module, preferably with a positive pressure of 0.06 MPa for 30 seconds, and then moves the double-layer plate back.

[0029] During sealing, the robotic arm controls the gripper to move the PVDF plate to the Waste plate of the heating and shaking module; the automated pipetting workstation automatically pipes the sealing reagent into each well, preferably 150 μl per well; shakes at room temperature and 500 rpm for 45 min; the robotic arm controls the gripper to move the PVDF plate back to the plate position of the waste liquid tank; the robotic arm controls the gripper to move the double-layer plate position to the corresponding position of the positive pressure module, preferably with a positive pressure of 0.06 MPa for 30 s, and then moves the double-layer plate back.

[0030] In a preferred embodiment of this application, the colloidal sample undergoes a second decolorization and a second solidification process before enzymatic hydrolysis. The purpose is to thoroughly clean and fix the colloidal sample after reduction and blocking, remove excess reagents, and prepare it for enzymatic hydrolysis. The specific steps are as follows: The automated pipetting workstation automatically pipes cleaning fluid into each well, preferably 900 μl per well. A robotic arm moves the grippers to the Waste plate of the heated and vibrating module, preferably at 25°C and 800 rpm for 5 minutes. The robotic arm then moves the grippers back to the waste tank position. Next, the robotic arm moves the grippers to the corresponding position of the double-layer plate, applying positive pressure of 0.06 MPa for 30 seconds, and then moves the double-layer plate back. This process is repeated twice for cleaning.

[0031] During the secondary decolorization, the automated pipetting workstation automatically pipes the decolorizing solution into each well, preferably 900 μl per well. The robotic arm moves the gripper to the Waste plate of the heated and vibrating module of the automated pipetting workstation, preferably vibrating at 25°C and 800 rpm for 10 min. The robotic arm then moves the gripper back to the waste liquid tank. The robotic arm then moves the gripper to the corresponding position of the double-layer plate, preferably applying a positive pressure of 0.06 MPa for 30 s, and moves the double-layer plate back. The above steps are repeated 3 times to perform secondary decolorization of the colloidal sample.

[0032] During the secondary curing process, the automated pipetting workstation automatically pipes the curing solution into each well, preferably 500 μl per well. The robotic arm moves the gripper to the Waste plate of the heated and vibrating module of the automated pipetting workstation, preferably vibrating at 25°C and 500 rpm for 5 minutes. The robotic arm then moves the gripper back to the waste liquid tank. The robotic arm then moves the gripper to the corresponding position of the double-layer plate, preferably applying a positive pressure of 0.06 MPa for 30 seconds, and moves the double-layer plate back. The above steps are repeated twice to perform secondary curing of the granule sample.

[0033] After the secondary curing of the granule sample is completed, the waste plate on the heating and vibration module is replaced with a collection plate after a delay. The robotic arm controls the gripper to move the PVDF plate to the collection plate of the heating and vibration module, forming a double-layer plate position.

[0034] The enzymes in the colloidal sample are hydrolyzed into peptides. The specific steps are as follows: an automated pipetting workstation automatically pipes the enzyme solution into each well, preferably 200 μl per well; heat and shake at 37°C and 500 rpm for 1-2 h; the preferred peptide concentration is between 0.1-50 µg / µL; the automated pipetting workstation automatically pipes the solution into each well, and shakes and mixes at 500 rpm for 30 s; a robotic arm moves the gripper to the position of the double-layer plate, reaches the corresponding position of the positive pressure module, preferably with a positive pressure of 0.06 MPa for 30 s, and then moves the double-layer plate back to the heating and shaking module.

[0035] The purpose of elution is to separate the peptide products generated within the colloidal particles during the enzymatic hydrolysis reaction from the PVDF membrane and colloidal matrix. During elution, an automated pipetting station automatically pipes the eluent into each well, preferably 200 μl per well; the mixture is then shaken at 1000 rpm for 10 min; a robotic arm moves the grippers to the corresponding position of the bilayer plate, preferably applying a positive pressure of 0.06 MPa for 30 s, and then moves the bilayer plate back to the heating and shaking module; the automated pipetting station automatically pipes the eluent into each well, preferably 100 μl per well, and the mixture is shaken at 1000 rpm for 10 min; the robotic arm moves the grippers to the corresponding position of the positive pressure module, preferably applying a positive pressure of 0.06 MPa for 30 s; the peptide samples in the collection plate are collected and dried in a vacuum concentrator for later use.

[0036] After elution, to further remove salt ions and impurities from the peptide sample and obtain a high-purity peptide sample, the peptide sample undergoes desalting and final purification. Specific operational steps include: Update the 8-channel pipette tip and place the 8-channel pipette tip and desalting column on the corresponding rack in the automated pipetting workstation; update the 1.2ml deep-well plate and label it as the sample receiving plate; transfer washing solution D, washing solution E, and elution solution F to the storage tank and place it on the corresponding plate position in the automated pipetting workstation; place the collection plate containing the dried peptide sample in the automated pipetting workstation; the automated pipetting workstation automatically pipettes, adding washing solution D to the collection plate, preferably 100-150μL per well, and pipetting to reconstitute the peptide sample, preferably 5-10 times; the automated pipetting workstation automatically pipettes, adding the peptide sample to the desalting column for loading; the robotic arm moves the gripper to the corresponding position of the double-layer plate, reaching the positive pressure module, preferably at 0.06MPa positive pressure for 30s.

[0037] The automated pipetting workstation automatically pipes cleaning fluid D into each well; the robotic arm controls the gripper to move the double-layer plate to the corresponding position of the positive pressure module, preferably with a positive pressure of 0.06MPa for 30s; the above steps are repeated twice.

[0038] The automated pipetting workstation automatically pipes cleaning fluid E into each well; the robotic arm controls the gripper to move the double-layer plate to the corresponding position of the positive pressure module, preferably with a positive pressure of 0.06MPa for 30s; the above steps are repeated twice.

[0039] The robotic arm controls the grippers to move the desalting column to the sample receiving plate; the automated pipetting station automatically pipes the solution, adding elution buffer F to each well; the robotic arm controls the grippers to move the double-layer plate to the corresponding position of the positive pressure module, preferably with a positive pressure of 0.06 MPa for 30 seconds; the above steps are repeated twice for final elution; the peptide samples in the sample receiving plate are collected and placed in a vacuum concentrator to dry for later use.

[0040] The peptide sample obtained in the above steps was reconstituted with 10 µL of 0.1% formic acid, and 1 µL was analyzed using a Bruker TimsTOF HT mass spectrometer; the identification results are as follows. Figure 3 As shown, the results indicate that this invention, based on an automated pipetting workstation and a 96-well plate, achieves large-scale automated in-gel enzymatic digestion peptide collection, increasing the throughput to a maximum of 96 samples per batch, significantly reducing sample processing time and batch effects. Furthermore, by adding an automated desalting step, the risk of column clogging due to residual gel particles is reduced. Finally, through optimized pretreatment, this invention significantly shortens the experimental cycle, completing the entire in-gel enzymatic digestion process for at least 96 samples within 5 hours, a reduction of nearly 10 times compared to existing technologies requiring at least 48 hours.

[0041] Another embodiment of this application provides a high-throughput in-gel enzymatic peptide enrichment kit for implementing the high-throughput in-gel enzymatic peptide enrichment method of the previous embodiment; the in-gel enzymatic peptide enrichment kit of this embodiment includes: The decolorizing solution is preferably 50% acetonitrile and 50mM ammonium bicarbonate, with an addition volume of 500-1000µL; The cleaning solution is preferably 50mM ammonium bicarbonate, with an addition volume of 500-1000µL; The curing solution is preferably 100% acetonitrile, and the added volume is 500-1000µL. The reducing agent is dithiothreitol or trichloroethyl phosphate, preferably 5-20 mM trichloroethyl phosphate; The alkylating agent is preferably 10-55 mM chloroacetamide; The enzymatic hydrolysate is trypsin or a mixture of trypsin and recombinant lysinase, preferably a mixture of trypsin and recombinant lysinase; The preferred stop solution is 10 v / v% trifluoroacetic acid; A desalting column, wherein the packing material of the desalting column is a reversed-phase chromatography packing material, preferably C18; Eluent 1 is preferably composed of 60% acetonitrile and 0.1% trifluoroacetic acid, with an added volume of 150-200 µL; Eluent 2 is preferably composed of 90% acetonitrile and 0.1% trifluoroacetic acid, with an added volume of 150-200 µL; The cleaning solution D is preferably 100 v / v% ethyl acetate and 0.2 v / v% trifluoroacetic acid, with an addition volume of 150-200 µL; Cleaning solution E is preferably 0.2 v / v% trifluoroacetic acid, with an addition volume of 150-200 µL; The eluent F is preferably 5 v / v% ammonium hydroxide and 80 v / v% acetonitrile, added in a volume of 100 µL.

[0042] In this embodiment, the kit is applicable not only to automated pipetting workstations but also to manual operations. When operating manually, using a 96-well plate and a 96-well plate adapter, combined with centrifugation, can achieve a high-efficiency, high-throughput enrichment effect.

[0043] As one embodiment of this application, the mass spectrometry-grade acetonitrile, water, and formic acid were purchased from Thermo Fisher Scientific, the trifluoroacetic acid was purchased from Sigma Aldrich, and the PDVF plate was purchased from Guangzhou Megbio Biotechnology Co., Ltd.

[0044] This high-throughput in-gel enzymatic peptide enrichment kit, combined with an automated enrichment method, significantly improves experimental throughput, enabling parallel, unmanned processing of dozens to hundreds of samples, thus meeting the high-throughput requirements of large clinical cohort studies. Secondly, it greatly enhances experimental repeatability and accuracy, with mechanical control eliminating inconsistencies caused by human operation, laying a solid foundation for generating reliable and comparable mass spectrometry data. Finally, it standardizes the process, promoting the integration and validation of data between laboratories.

[0045] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An automated high-throughput intragel enzymatic peptide enrichment method, wherein the high-throughput intragel enzymatic peptide enrichment method employs a high-throughput tissue homogenizer and an automated pipetting workstation; characterized in that, The high-throughput intragel enzymatic peptide enrichment method includes the following steps: Grind the adhesive strip samples to obtain adhesive particle samples, and prepare the reagents for the automated pipetting workstation. The reagents are automatically added using an automated pipetting workstation, and the colloidal sample is sequentially decolorized, washed, solidified, reduced, blocked, enzymatically digested, and eluted to obtain peptide samples. Remove salt ions and impurities from the peptide sample and place it in a vacuum concentrator to dry it for later use.

2. The automated high-throughput intragel enzymatic peptide enrichment method according to claim 1, characterized in that, The automated pipetting workstation is equipped with: an operating table, a robotic arm with grippers, and two 8-channel pipettes; the operating table of the automated pipetting workstation contains: a trash can, a carrier with desalting columns, a carrier with 96-well pipette tips, a positive pressure module, two heating and shaking modules, a 96-well plate with PVDF membrane columns, two 1.2ml deep-well plates, one four-channel groove, and several plate positions; Among them, the 96-well plate containing the PVDF membrane column was labeled as PDVF plate; the two 1.2ml deep-well plates were labeled as Waste plate and Collection plate, respectively; and the four grooves were labeled as waste liquid groove. The reagents include: decolorizing solution, cleaning solution, curing solution, reducing reagent, alkylating reagent, enzymatic hydrolysate, stop solution, elution solution 1, elution solution 2, cleaning solution D, cleaning solution E, and elution solution F.

3. The automated high-throughput intragel enzymatic peptide enrichment method according to claim 2, characterized in that, The specific steps for grinding the adhesive strip sample to obtain the adhesive particle sample are as follows: Transfer the gel strip sample to a 2ml grinding tube, add grinding beads and destaining solution; place the grinding tube in a high-throughput tissue homogenizer for grinding, then remove the grinding beads to obtain the gel particle sample; Place the colloidal sample in a PDVF plate and then place it together in the plate slot containing the waste liquid tank.

4. The automated high-throughput intragel enzymatic peptide enrichment method according to claim 2, characterized in that, The specific steps for decolorizing and cleaning the colloidal sample are as follows: During decolorization, add decolorizing solution, shake and apply positive pressure, repeat 3 times; During cleaning, add cleaning solution, shake and apply positive pressure, repeat twice.

5. The automated high-throughput intragel enzymatic hydrolysis peptide enrichment method according to claim 2, characterized in that, The specific steps for curing the colloidal particle sample are as follows: After adding the curing liquid, shake at 500 rpm for 5 minutes, apply positive pressure of 0.06 MPa for 30 seconds, and repeat twice.

6. The automated high-throughput intragel enzymatic peptide enrichment method according to claim 2, characterized in that, The specific steps for restoring and sealing the colloidal sample are as follows: When breaking the disulfide bonds in the colloidal sample, after adding the reducing reagent, shake at 1000 rpm for 1 hour and apply a positive pressure of 0.06 MPa for 30 seconds. When blocking the thiol groups of the colloidal sample, after adding the alkylating agent, shake at 500 rpm for 45 min and apply a positive pressure of 0.06 MPa for 30 s.

7. The automated high-throughput intragel enzymatic peptide enrichment method according to claim 6, characterized in that, Before enzymatic hydrolysis, the colloidal sample undergoes a second decolorization and a second curing process. The specific steps are as follows: After adding the cleaning solution, shake at 800 rpm for 5 minutes, apply positive pressure of 0.06 MPa for 30 seconds, and repeat twice. For the second decolorization, after adding the decolorizing solution, shake at 800 rpm for 10 min, apply positive pressure of 0.06 MPa for 30 s, and repeat 3 times. During the second curing process, after adding the curing liquid, shake at 500 rpm for 5 minutes, apply a positive pressure of 0.06 MPa for 30 seconds, and repeat twice.

8. The automated high-throughput intragel enzymatic peptide enrichment method according to claim 2, characterized in that, The specific steps for enzymatic digestion of colloidal samples are as follows: After adding the enzyme hydrolysate, shake at 500 rpm for 1-2 hours; After adding the stop solution, shake at 500 rpm for 30 seconds and apply a positive pressure of 0.06 MPa for 30 seconds.

9. The automated high-throughput intragel enzymatic peptide enrichment method according to claim 2, characterized in that, The specific steps for eluting the colloidal sample are as follows: After adding elution buffer 1, shake at 1000 rpm for 10 min, and apply positive pressure of 0.06 MPa for 30 s; After adding eluent 2, shake at 1000 rpm for 10 min, and apply positive pressure of 0.06 MPa for 30 s; The specific steps for removing salt ions and impurities from peptide samples are as follows: After reconstitution of the peptide sample with 0.1% FA, it was added to a desalting column for treatment at 0.06 MPa positive pressure for 30 seconds. After adding cleaning solution D, apply a positive pressure of 0.06 MPa for 30 seconds, and repeat twice. After adding cleaning fluid E, apply a positive pressure of 0.06 MPa for 30 seconds, and repeat twice. After adding elution buffer F, apply a positive pressure of 0.06 MPa for 30 seconds, and repeat twice.

10. A high-throughput in-gel enzymatic peptide enrichment kit, used to implement the high-throughput in-gel enzymatic peptide enrichment method as described in claim 2, characterized in that, The automated high-throughput in-gel enzymatic peptide enrichment kit includes: The decolorizing solution is preferably composed of 50% acetonitrile and 50mM ammonium bicarbonate; The preferred cleaning solution is 50 mM ammonium bicarbonate. The curing solution is preferably 100% acetonitrile; The reducing agent is dithiothreitol or trichloroethyl phosphate, preferably 5-20 mM trichloroethyl phosphate; The alkylating agent is preferably 10-55 mM chloroacetamide; The enzymatic hydrolysate is trypsin or a mixture of trypsin and recombinant lysinase, preferably a mixture of trypsin and recombinant lysinase; The preferred stop solution is 10 v / v% trifluoroacetic acid; A desalting column, wherein the packing material of the desalting column is a reversed-phase chromatography packing material, preferably C18; Eluent 1 is preferably composed of 60% acetonitrile and 0.1% trifluoroacetic acid; Eluent 2 is preferably composed of 90% acetonitrile and 0.1% trifluoroacetic acid; Cleaning solution D is preferably 100 v / v% ethyl acetate and 0.2 v / v% trifluoroacetic acid; Cleaning solution E is preferably 0.2 v / v% trifluoroacetic acid; The eluent F is preferably 5 v / v% ammonium hydroxide and 80 v / v% acetonitrile.

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