Continuous batch pretreatment device and method for proteomics, glycoproteomics and phosphorylated proteomics samples

Abstract

The invention relates to the field of material analytical chemistry and biochemistry, and provides a continuous batch pretreatment device and method for proteomics, glycoproteomics and phosphorylated proteomics samples. According to the continuous batch pretreatment device for the proteomics, glycoproteomics and phosphorylated proteomics samples, under the assistance of the filler, the whole process of protein denaturation, aggregation capture, enzymolysis and complete glycopeptide and phosphorylated peptide enrichment in biological samples can be achieved. Compared with a free solution method conventionally used in proteomics, the sample pretreatment method based on the device has the advantages that the sample treatment time can be effectively shortened, and continuous analysis of mass proteome, glycoprotein group and phosphorylated proteome of trace protein samples can be realized; the method is expected to be widely applied to the aspects of disease occurrence mechanisms, drug action mechanisms, clinical detection, biomarker discovery and the like.

Description

Technical Field

[0001] This invention relates to the fields of materials analytical chemistry and biochemistry, and in particular to a continuous batch pretreatment apparatus and method for proteomics, glycoproteomics and phosphorylated proteomics samples. Background Technology

[0002] Proteomics and post-translational modification (PTM) proteomics, as a technological system with practical application value, are finding increasingly diverse applications in life process exploration, disease mechanism analysis, and biomarker discovery. Recently, multi-omics-driven precision oncology based on proteomics and PTM proteomics has become a hot topic and development trend for clinical applications. While multi-omics proteomics technologies are extremely important, the various post-translational modifications significantly increase the complexity of proteomic research, particularly posing greater challenges to sample pretreatment techniques: 1. For samples with significant batch and individual variability, preciousness, and trace amounts, highly reproducible and robust methods need to be developed; 2. Given the wide dynamic range of proteins in biological samples, methods with in-depth protein identification capabilities and high protein species coverage need to be developed.

[0003] Existing research indicates that multi-omics analysis at the protein level (proteomics and post-translational modification proteomics) typically requires independent, sequential, and parallel mass spectrometry analysis of both proteomics and post-translational modification proteomics samples. While this independent and parallel approach can yield important biological insights, it demands large initial sample volumes and multiple batches of sample preprocessing, leading to a waste of valuable clinical samples. Furthermore, the separate processing of proteomics and post-translational modification proteomics samples reduces the correlation between the two. Therefore, developing a cascaded approach to achieve one-stop, integrated, and efficient multi-omics research on various biological samples, even with limited or trace amounts of biological samples, to generate more comprehensive multi-omics datasets, will greatly advance further in-depth research.

[0004] Currently, integrated, one-stop sample pretreatment platforms for single-level proteomics are constantly being developed. These technologies generally include steps such as protein capture, online enzymatic digestion, and desalting, significantly improving the sensitivity, stability, and throughput of sample pretreatment. Among them, Tian Ruijun et al. developed an integrated proteomics pretreatment technology based on centrifugal pipette tips. This technology uses ion exchange materials to capture proteins, followed by rapid enzymatic digestion and C18 desalting, achieving a one-stop protein pretreatment process from the original sample. Mann et al. developed the iST technology based on sample processing in a closed pipette tip. The entire operation is carried out in a closed pipette tip equipped with a C18 pad, which can reduce sample contamination and loss. Jeroen et al. developed the SP3 method based on the hydrophilic interaction between magnetic beads and proteins. This method uses ethanol to drive the capture of proteins on the surface of hydrophilic magnetic beads, and removes impurities such as surfactants and solvents by rinsing.

[0005] Although these technologies and integrated platforms have broad application prospects in different sample types (including body fluids, tissues and cells), they are only suitable for single proteomics studies and cannot simultaneously obtain post-translational modification proteomics information, let alone depict a panoramic view of multi-omics proteomics.

[0006] Therefore, there is an urgent need to develop an integrated technology platform that can perform highly reproducible and in-depth coverage analysis of the panoramic proteome and post-translational modification proteome (such as phosphorylation and glycosylation) from the same biological sample. This device and related methods should not only promote the effective integration of proteomics data, but also improve the utilization rate of valuable clinical samples. Summary of the Invention

[0007] To construct an integrated proteomics research platform, this invention first develops a universal protein capture strategy tailored to the characteristics of clinical tissue and blood sample types, reducing the initial sample volume required. Second, it designs and examines the processing efficiency of different enzyme digestion methods and parameters, screening for optimal parameters to improve sample utilization. Third, it integrates glycopeptide and phosphorylated peptide enrichment strategies developed in previous work, establishing enrichment workflows for single-omics (proteomics, glycoproteomics, phosphorylated proteomics) and multi-omics (serial enrichment) research purposes. The parameter properties of each step are examined, and the various workflows of the integrated platform are continuously improved. Ultimately, this results in a one-stop, integrated proteomics pretreatment platform capable of continuous and in-depth analysis of the panoramic proteome and post-translational modification proteome (such as phosphorylation and glycosylation) from trace amounts of clinical samples.

[0008] More specifically, in order to address the above-mentioned needs, this invention proposes a continuous batch pretreatment device and method for proteomics, glycoproteomics, and phosphorylated proteomics samples. The aim is to achieve one-stop, integrated, and efficient proteomics research on various biological samples under limited or even trace biological sample conditions by using a cascaded approach, thereby generating a more comprehensive proteomics dataset.

[0009] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a continuous batch pretreatment device for proteomics, glycoproteomics, and phosphorylated proteomics samples. The device includes: centrifuge tubes or well plates, and packing materials dispersed in the centrifuge tubes or well plates that can achieve protein aggregation, glycopeptide enrichment, and phosphorylated peptide enrichment.

[0010] In the continuous batch sample pretreatment device for proteomics, glycoproteomics, and phosphorylated proteomics provided by this invention, the centrifuge tubes are 0.5-1.5 mL in size, and the well plates are any one of 6, 12, 24, 48, 96, or 384-well plates; and / or, the packing material dispersed in the centrifuge tubes that enables protein aggregation, glycopeptide enrichment, and phosphorylated peptide enrichment includes: One or a mixture of two packing materials selected from hydrophilic interaction chromatography (HILIC) materials, metal oxide affinity chromatography (MOAC) materials, immobilized metal affinity chromatography (IMAC) materials, ion exchange materials, and magnetic materials; magnetic materials include, but are not limited to, commercially available magnetic nano or micro materials and self-made magnetic nano or micro materials.

[0011] The filling amount of the one or two fillers mixture is 0.5-10 mg; and / or, the mass ratio of the two fillers in the filler mixture is 1:(1-10). Preferably, the hydrophilic interaction chromatographic materials include, but are not limited to, commercially available ZIC-HILIC, amino silica gel, Click-Mal materials, and Click-Cys materials; the metal oxide affinity chromatographic materials include, but are not limited to, TiO2 and ZrO2; and the magnetic materials include, but are not limited to, commercially available magnetic nano or micro materials and self-made magnetic nano or micro materials.

[0012] The filler is one or a mixture of two of the following: HILIC filler, titanium dioxide material, IMAC material, ion exchange material, and magnetic material; and / or, the filling amount of the filler is 0.5-10 mg; and / or, the mass ratio of the two filler mixtures is 1:(1-5).

[0013] Secondly, the present invention provides a method for continuous batch pretreatment of samples for continuous proteomics, glycoproteomics, and phosphorylated proteomics, comprising: (1) Dissolve the protein sample to be tested in the first reagent and add it to the above-mentioned continuous batch pretreatment device for proteomics, glycoproteomics and phosphorylated proteomics samples. After heating treatment, add the second reagent and carry out the reaction in the dark. (2) Add one or two filler mixtures and a third reagent respectively to make the protein sample after reaction aggregate onto one or two filler mixtures, and wash with a fourth reagent to remove the washing solution; And / or, if the packing material is magnetic, a matching magnetic separator is required to separate the material from the solution and remove the cleaning solution; (3) After the fifth reagent is added to fully dissolve the protein, the sixth reagent is added to dilute the sample, and the protease solution is added to complete the enzymatic hydrolysis process to obtain the enzymatically hydrolyzed polypeptide sample; (4) Add the seventh reagent to the centrifuge tube, adjust the enzymatic hydrolysis peptide sample system to the glycopeptide loading solvent system and carry out the glycopeptide enrichment process. The eluent is evaporated and used for glycoproteome analysis. At the same time, the loading solution and the rinsing solution during the enrichment process are collected and combined. And / or, if the filler is a magnetic material, a matching magnetic separator is required to separate the magnetic material adsorbing glycopeptides from the solution. In this case, the magnetic material adsorbs glycopeptides.

[0014] (5) After the combined glycopeptide loading solution and eluent are evaporated, the eighth reagent is added to adjust the solvent system to the phosphorylated peptide loading solvent system and carry out the phosphorylated peptide enrichment process. The eluent is evaporated for phosphorylated proteomics analysis. At the same time, the phosphorylated peptide loading solution and eluent during the enrichment process are collected and combined. And / or, the packing material is a magnetic material. A matching magnetic separator is required to separate the magnetic material adsorbing phosphorylated peptides from the solution. At this time, the magnetic material adsorbs phosphorylated peptides. (6) The combined phosphorylated peptide loading solution and eluent were evaporated to dryness, then desalted by C18 and used for proteomics analysis. The filler material is a magnetic material, and a matching magnetic separator is required to separate the material from the solution. At this time, the non-glycopeptide and non-phosphorylated peptide portions adsorbed on the magnetic material are used for the identification and analysis of proteomic results.

[0015] In the method provided by the present invention, the mass of the protein sample to be tested is 20 μg-5 mg.

[0016] In the method provided by the present invention, the first reagent is an ammonium bicarbonate solution containing dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine hydrochloride (TCEP); The second reagent is an ammonium bicarbonate solution containing iodoacetamide or iodoacetic acid; The third reagent is a mixture of one or more organic reagents; The fourth reagent is a mixed aqueous solvent of one or more organic reagents; Alternatively, the third reagent is one or more of acetonitrile, ethanol, or methanol; the fourth reagent is one or more of acetonitrile, ethanol, or methanol in an aqueous solution.

[0017] In the method provided by the present invention, the fifth reagent is an aqueous solution containing urea or a surfactant; the sixth reagent is an ammonium bicarbonate solution.

[0018] In the method provided by this invention, the seventh reagent is an organic reagent, or the seventh reagent is acetonitrile, ethanol, or methanol.

[0019] In the method provided by this invention, the eighth reagent is an organic solvent containing glycolic acid or trifluoroacetic acid.

[0020] In the method provided by the present invention, the temperature of the heat treatment is 90-100℃.

[0021] In the method provided by the present invention, the packing material or the mixture of two packing materials is one or two of the following: hydrophilic interaction chromatographic materials, metal oxide affinity chromatographic materials, immobilized metal affinity chromatographic materials, ion exchange materials, and magnetic materials. Preferably, the HILIC material includes, but is not limited to, commercially available ZIC-HILIC, amino silicone, Click-Mal, and Click-Cys materials; the metal oxide affinity chromatography material includes, but is not limited to, TiO2 and ZrO2; and the magnetic material includes, but is not limited to, commercially available magnetic nanomaterials or micromaterials and self-made magnetic nanomaterials or micromaterials.

[0022] The beneficial effects of this invention are as follows: The integrated sample pretreatment device for continuous proteomics, glycoproteomics, and phosphorylated proteomics provided by this invention disperses the packing material that enables protein aggregation, glycopeptide enrichment, and phosphorylated peptide enrichment in centrifuge tubes. Within the centrifuge tubes, continuous protein aggregation, glycopeptide enrichment, and phosphorylated peptide enrichment and separation can be achieved. Under limited or even trace biological sample conditions, a one-stop, integrated, and efficient multi-omics proteomics study can be carried out on various biological samples using a tandem approach, generating a more comprehensive multi-omics proteomics dataset. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the integrated device for sample pretreatment of continuous proteomics, glycoproteomics, and phosphorylated proteomics according to the present invention.

[0025] Figure 2 This invention illustrates the effect of different enzymatic hydrolysis times and different amounts of enzyme used on the identification results.

[0026] Figure 3 This is a comparison of the effects of the present invention with traditional sample pretreatment methods in free solution conditions.

[0027] Figure 4 The results of glycopeptide identification in different biological samples are presented in this invention.

[0028] Figure 5 The results of proteomics, glycoproteomics, and phosphorylated proteomics identifications performed on different biological samples in this invention are presented. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0030] The following describes the continuous batch sample pretreatment apparatus for proteomics, glycoproteomics, and phosphorylated proteomics provided by this invention, such as... Figure 1 The device includes centrifuge tubes and packing materials dispersed within the tubes, enabling continuous protein aggregation, glycopeptide enrichment, and phosphorylated peptide enrichment. The sample pretreatment apparatus and method provided in this embodiment aim to achieve one-stop, integrated, and efficient proteomics research on various biological samples under limited or even trace biological sample conditions using a cascaded approach. This effectively avoids sample loss and cross-contamination caused by multiple sample transfers and shortens the sample pretreatment steps, which originally required 2-3 days, to one day, effectively improving the efficiency of proteomics work.

[0031] In some embodiments, the continuous proteomics, glycoproteomics, and phosphorylated proteomics sample pretreatment can achieve sample pretreatment for all three omics, or it can achieve sample pretreatment for one or two omics, but is not limited thereto.

[0032] The English abbreviations and Chinese translations of the reagents used in this invention are as follows: DTT: Dithiothreitol.

[0033] IAA: Iodoacetamide.

[0034] TFA: Trifluoroacetic acid.

[0035] ACN: Acetonitrile.

[0036] FA: Formic acid.

[0037] TiO2: Titanium dioxide.

[0038] Example 1

[0039] This embodiment provides a method for continuous batch pretreatment of proteomics, glycoproteomics, and phosphorylated proteomics samples, the steps of which are as follows: 1. Take 100 μg of protein sample into a 1.5 mL centrifuge tube, dilute with water to 1 mg / mL, add 200 mM DTT (dissolved in 50 mM ammonium bicarbonate solution) to a final concentration of about 10 mM, and heat at 95℃ for 10 min.

[0040] 2. Continue adding 800 mM IAA (dissolved in 50 mM ammonium bicarbonate solution) to the centrifuge tube until the final concentration is about 40 mM, and react in the dark for 30 min.

[0041] 3. Continue adding 3 mg of hydrophilic interaction chromatographic material (dispersed in 60 μL of water) to the centrifuge tube, and add acetonitrile to a final concentration of 70%. Vortex to mix, let stand for 10 min, and centrifuge at 8000 rpm for 5 min. Wash three times with 80% ethanol for protein capture.

[0042] 4. Continue to add 32 μL of 8M urea (dissolved in 50 mM ammonium bicarbonate solution) to the centrifuge tube, sonicate until dissolved, add 224 μL of 50 mM ammonium bicarbonate solution, add Trypsin, and enzymatically digest at 37°C for 2 hours.

[0043] 5. Continue adding 1094 μL of acetonitrile and 14 μL of TFA to the centrifuge tube.

[0044] 6. Then, perform oscillation and adsorption for 10 min. Centrifuge, transfer the supernatant to a new centrifuge tube, and evaporate to dryness for later use.

[0045] 7. After centrifugation at the bottom, add 50 μL of 80% ACN / 1% TFA to the material, rinse and shake 4 times, centrifuge, and collect the supernatant after rinsing.

[0046] 8. The collected supernatant was eluted three times with 30% ACN / 1% FA 30 μL to obtain glycopeptides.

[0047] 9. Continue adding TiO2 to the centrifuge tube to enrich phosphorylated peptides (mix the glycopeptide loading solution and eluent and evaporate to dryness).

[0048] 10. Add 50 μL of 80% ACN / 5% TFA / 1 M glycolic acid to the centrifuge tube to equilibrate TiO2.

[0049] 11. Add 100 μL of 80% ACN / 5% TFA / 1 M glycolic acid to a centrifuge tube to reconstitute the sample.

[0050] 12. Add 50 μL each of 80% ACN / 5% TFA / 1 M glycolic acid, 80% ACN / 1% FA, 20% ACN / 0.1% FA, and 20% ACN to the centrifuge tubes and rinse them separately.

[0051] 13. Add 10% ammonia water to the centrifuge tube to wash off the impurities, then rotate dry and store.

[0052] 14. Add C18 material to the elution buffer of phosphorylated peptides to the centrifuge tube to desalt them, and then proceed with mass spectrometry detection.

[0053] Example 2

[0054] The only difference between Example 2 and Example 1 is the use of multi-well plates for continuous batch pretreatment of proteomics, glycoproteomics, and phosphorylated proteomics samples. The centrifugation process employs a centrifuge or magnetic separator compatible with the multi-well plate method.

[0055] Example 3

[0056] The only difference between Example 3 and Example 1 is the use of magnetic materials and magnetic separators for continuous batch pretreatment of proteomics, glycoproteomics, and phosphorylated proteomics samples. The magnetic material can be BeaverBeads™ MagCOOH magnetic beads (1μm, 10mg / mL, 5mL), where proteins are aggregated on the surface of the magnetic beads to begin subsequent sample pretreatment operations.

[0057] Example 4

[0058] The only difference between Example 4 and Example 1 is the time used in the enzymatic hydrolysis process. Here, the effects of 0.5 hours, 1 hour, 2 hours, 2.5 hours, 3 hours, and overnight incubation, as well as different enzyme dosages, on the identification results were examined. Other conditions were handled according to the general conditions described in Example 1, and the protein identification effects were compared. The results are as follows... Figure 2 As shown, a hydrolysis time of 0.5 hours and an enzyme amount of E:S = 1:50 are sufficient to effectively achieve proteolytic hydrolysis. Therefore, the device and related methods provided by this invention can effectively improve work efficiency.

[0059] Example 5

[0060] The only difference between Example 5 and Example 1 is the comparison of their effects with traditional sample pretreatment methods in a free solution state. The results are as follows: Figure 3 As shown, the method provided by the present invention has better protein identification effect than traditional sample pretreatment methods in free solution state, thus proving that the device and related methods provided by the present invention can effectively improve work efficiency.

[0061] Example 6

[0062] The only difference between Example 6 and Example 1 is that the effects were compared using different trace biological samples, and the results are as follows: Figure 4 As shown, the method provided by the present invention can successfully perform sample pretreatment and identify glycopeptides in different biological samples, proving the universality of the device and method provided by the present invention.

[0063] Example 7

[0064] The only difference between Example 7 and Example 1 is that magnetic nanomaterials and different biological samples were used to verify the effect, and the results are as follows: Figure 5 As shown, the method provided by this invention can successfully use magnetic nanomaterials to pretreat samples in different biological samples and complete the identification of proteomics, glycoproteomics, and phosphorylated proteomics, which once again proves the universality of the device and method provided by this invention.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A continuous batch sample pretreatment device for proteomics, glycoproteomics, and phosphorylated proteomics, characterized in that, The device includes: centrifuge tubes or well plates, and packing material dispersed in the centrifuge tubes or well plates that enables protein aggregation, glycopeptide enrichment, and phosphorylated peptide enrichment.

2. The continuous batch sample pretreatment device for proteomics, glycoproteomics, and phosphorylated proteomics according to claim 1, characterized in that, The centrifuge tubes are 0.5-1.5 mL in size, and the well plates are any one of 6, 12, 24, 48, 96, or 384-well plates; and / or, the packing material dispersed in the centrifuge tubes capable of protein aggregation, glycopeptide enrichment, and phosphorylated peptide enrichment includes one or a mixture of multiple packing materials selected from hydrophilic interaction chromatographic materials, metal oxide affinity chromatographic materials, immobilized metal affinity chromatographic materials, ion exchange materials, and magnetic materials; the loading amount of the one or more packing materials is 0.5-10 mg; and / or, the mass ratio of the two packing materials in the packing mixture is 1:(1-10). Preferably, the hydrophilic interaction chromatographic materials include, but are not limited to, commercially available ZIC-HILIC, amino silica gel, Click-Mal materials, and Click-Cys materials; the metal oxide affinity chromatographic materials include, but are not limited to, TiO2 and ZrO2; and the magnetic materials include, but are not limited to, commercially available magnetic nano or micro materials and self-made magnetic nano or micro materials.

3. A method for continuous batch pretreatment of samples for continuous proteomics, glycoproteomics, and phosphorylated proteomics, characterized in that, include: (1) Dissolve the protein sample to be tested in the first reagent and add it to the continuous batch pretreatment device for proteomics, glycoproteomics and phosphorylated proteomics samples as described in any one of claims 1-2. After heating treatment, add the second reagent and carry out a light-protected reaction. (2) Add one or two filler mixtures and a third reagent respectively to make the protein sample after reaction aggregate onto one or two filler mixtures, and wash with a fourth reagent to remove the washing solution; And / or, if the packing material is magnetic, a matching magnetic separator is required to separate the material from the solution and remove the cleaning solution; (3) After the fifth reagent is added to fully dissolve the protein, the sixth reagent is added to dilute the sample, and the protease solution is added to complete the enzymatic hydrolysis process to obtain the enzymatically hydrolyzed polypeptide sample; (4) Add the seventh reagent to the centrifuge tube, adjust the enzymatic hydrolysis peptide sample system to the glycopeptide loading solvent system and carry out the glycopeptide enrichment process. The eluent is evaporated and used for glycoproteome analysis. At the same time, the loading solution and the rinsing solution during the enrichment process are collected and combined. And / or, the filler is a magnetic material, and a matching magnetic separator is used to separate the magnetic material adsorbing glycopeptides from the solution, at which point the magnetic material adsorbs glycopeptides. (5) After the combined glycopeptide loading solution and eluent are evaporated, the eighth reagent is added to adjust the solvent system to the phosphorylated peptide loading solvent system and carry out the phosphorylated peptide enrichment process. The eluent is evaporated for phosphorylated proteome analysis. At the same time, the phosphorylated peptide loading solution and eluent during the enrichment process are collected and combined. And / or, the filler is a magnetic material, and a matching magnetic separator is used to separate the magnetic material adsorbing phosphorylated peptides from the solution, at which point the magnetic material adsorbs phosphorylated peptides. (6) The combined phosphorylated peptide loading solution and eluent were evaporated to dryness, then desalted by C18 and used for proteomics analysis. And / or, the filler is a magnetic material, and a matching magnetic separator is used to separate the material from the solution. In this case, the adsorbed part of the magnetic material is a polypeptide of non-glycopeptide and non-phosphorylated peptide, which is used for the identification and analysis of proteomic results.

4. The method according to claim 3, characterized in that, The mass of the protein sample to be tested was 20 μg-5 mg.

5. The method according to claim 3, characterized in that, The first reagent is an ammonium bicarbonate solution containing dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine hydrochloride (TCEP); The second reagent is an ammonium bicarbonate solution containing iodoacetamide or iodoacetic acid; The third reagent is a mixture of one or more organic reagents; The fourth reagent is a mixed aqueous solvent of one or more organic reagents; Alternatively, the third reagent is one or more of acetonitrile, ethanol, or methanol; the fourth reagent is one or more of acetonitrile, ethanol, or methanol in an aqueous solution.

6. The method according to claim 3, characterized in that, The fifth reagent is an aqueous solution containing urea or a surfactant; the sixth reagent is an ammonium bicarbonate solution.

7. The method according to claim 3, characterized in that, The seventh reagent is an organic reagent, or the seventh reagent is acetonitrile, ethanol, or methanol.

8. The method according to claim 3, characterized in that, The eighth reagent is an organic solvent containing glycolic acid or trifluoroacetic acid.

9. The method according to claim 3, characterized in that, The temperature of the heat treatment is 90-100℃.

10. The method according to claim 3, characterized in that, The packing material or mixture of two packing materials mentioned above is one or a mixture of two packing materials selected from hydrophilic interaction chromatographic materials, metal oxide affinity chromatographic materials, immobilized metal affinity chromatographic materials, ion exchange materials, and magnetic materials. Preferably, the HILIC material includes, but is not limited to, commercially available ZIC-HILIC, amino silica gel, Click-Mal material, and Click-Cys material; the metal oxide affinity chromatography material includes, but is not limited to, TiO2 and ZrO2; Magnetic materials include, but are not limited to, commercially available magnetic nanomaterials or micromaterials, and homemade magnetic nanomaterials or micromaterials.

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