A rapid and high-throughput method for continuous enrichment of multiple protein post-translational modifications

Abstract

The present invention relates to a method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, which includes adding a lysate containing SDS mass concentration (g / ml) of 0.5% to 10% to a biological sample to obtain a crude protein extract; then adding a cyclodextrin or cyclodextrin derivative solution to mix, forming an SDS-cyclodextrin complex and then enzymatically hydrolyzing to obtain a polypeptide solution; first using a post-translational modification antibody to enrich ubiquitinated and / or acetylated modified peptides in a polypeptide solution, centrifuging, washing, eluting, and obtaining ubiquitinated modified polypeptide enrichment solution and / or acetylated modified polypeptide enrichment solution; then directly adding ACN and TFA to the supernatant after antibody enrichment to adjust the pH of the supernatant, and using enrichment materials to enrich phosphorylated modified peptides and / or glycosylated modified peptides. This method can enrich target modified peptides using relevant enrichment materials without desalting in the process of enriching different post-translational modified polypeptides, which can significantly shorten the time required for enrichment and improve detection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a method for rapid high-throughput continuous enrichment of post-translational modifications of multiple proteins. Background Art

[0002] Protein post-translational modifications (PTMs) are the addition or removal of specific groups on the amino acid residues of proteins, thereby regulating protein activity, localization, expression, and interaction with other cellular molecules. They are key factors in increasing the functional diversity of the proteome. These post-translational modifications include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, etc. The most common ones are ubiquitination, acetylation, phosphorylation, glycosylation, etc. In the process of identifying post-translational modifications of proteins, the protein will first be cleaved into peptides and then enter the mass spectrometry for analysis. However, due to the low abundance of post-translationally modified proteins in the sample, it is usually necessary to enrich the target modified peptides before mass spectrometry analysis.

[0003] CN103940894A A method for simultaneously enriching phosphorylated peptides and glycosylated peptides and performing mass spectrometry analysis, the method utilizes the electrostatic interaction between the positively charged amino groups on the surface of magnetic nanomaterials and the negatively charged phosphate peptides to achieve the selective enrichment of phosphorylated peptides, and then utilizes the hydrophilicity of the amino groups on the surface of magnetic nanomaterials to achieve the selective enrichment of glycosylated peptides, but it is only compatible with the enrichment of phosphorylated peptides and glycosylated peptides, and has high requirements for the purity of the peptide solution sample. P Mertins et al. published "Integrated proteomic analysis of post-translational modifications by serial enrichment", which can continuously enrich phosphorylated, ubiquitinated and acetylated modified peptides, but the method requires desalting and purification of the peptide solution before enrichment, and desalting in each enrichment step is time-consuming and easily causes the loss of some modified peptides such as glycosylated peptides, making it difficult to achieve high-throughput enrichment of multiple modified peptides such as phosphorylation, ubiquitination, acetylation and glycosylation, and the starting sample of the method is a purified peptide freeze-dried powder.

[0004] Moreover, due to the low abundance of post-translationally modified proteins and peptides, enrichment experiments often require a larger sample starting amount. For example, 1 mg of protein is usually used for each sample of various modifications. To achieve multiple modification enrichment, 5-10 mg of protein is generally required. However, the properties of various modified peptides are different and the solution systems in the enrichment process are quite different. When analyzing multiple post-translation modifications of the same sample, multiple desalting or solution replacement is often required during the processing to ensure a higher sample starting amount. Each desalting process loses at least 10% of the post-translation modified peptides, and the required desalting time is long, the operation steps are cumbersome, and the repeatability is poor. Therefore, studying a method for rapid, high-throughput, and continuous enrichment of multiple protein post-translation modifications is of great significance in revealing the functional regulatory mechanism of protein modifications and their role in biological processes. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a method for rapid and high-throughput continuous enrichment of multiple protein post-translational modifications, the purpose of which is to find that after a biological sample is lysed with a lysis solution containing SDS to obtain a crude protein extract, cyclodextrin or a cyclodextrin derivative is added to form an SDS-cyclodextrin complex with SDS, which can not only avoid the influence of SDS on the enrichment of post-translationally modified peptides by antibodies, but also enrich the target modified peptides using relevant enrichment materials without desalting treatment during the enrichment of different post-translationally modified peptides, thereby solving the technical problems that the existing continuous preparation of proteomics samples with multiple post-translation modifications requires multiple desalting and purification treatments, which easily results in a large loss of post-translationally modified peptides, a long desalting time, cumbersome operation steps, and poor repeatability.

[0006] To achieve the above object, according to one aspect of the present invention, a method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications is provided, which comprises the following steps:

[0007] (1) Extracting proteins: adding a lysis buffer containing 0.5% to 10% SDS mass concentration (g / ml) to the biological sample to obtain a crude protein extract;

[0008] (2) proteolysis: adding cyclodextrin or cyclodextrin derivative solution to the crude protein extract obtained in (1) and mixing well to form an SDS-cyclodextrin complex, followed by enzymatic hydrolysis to obtain a polypeptide solution;

[0009] (3) Continuously enriching multiple post-translational modifications of proteins: first, using a post-translational modification antibody to enrich the ubiquitinated and / or acetylated modified peptides in the polypeptide solution in (2), centrifuging, washing, and eluting to obtain a ubiquitinated modified polypeptide enrichment solution and / or an acetylated modified polypeptide enrichment solution;

[0010] ACN and TFA are then directly added to the supernatant after antibody enrichment to adjust the pH of the supernatant, and the enrichment material is used to enrich the phosphorylated modified peptides and / or glycosylated modified peptides.

[0011] Preferably, in the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, the enrichment material for enriching phosphorylated modified peptides is IMAC, the pH of the supernatant is adjusted to 3-4, and / or the enrichment material for enriching phosphorylated modified peptides is metal oxide, the pH of the supernatant is adjusted to 0.05-0.15, and the metal oxide includes TiO2 and CaTiO3.

[0012] Preferably, the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications uses IMAC-CaTiO3 to enrich phosphorylated modified peptides in series, and collects the enriched eluates twice.

[0013] Preferably, the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications first adds an equal volume of 1% TFA / ACN to the antibody enriched supernatant, and then uses Fe 3+ -IMAC was used to enrich phosphorylated peptides, and the supernatant was collected by centrifugation. ACN and TFA were added to the supernatant to adjust its pH to 0.8-0.1. CaTiO3 was used to enrich phosphorylated peptides again, and the eluents enriched twice were collected.

[0014] Preferably, in the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, the enrichment material for enriching glycosylated modified peptides is IMAC, and the IMAC comprises Ti 4+ -IMAC.

[0015] Preferably, the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications first uses enrichment materials to enrich phosphorylated modified peptides, and then enriches glycosylated modified peptides.

[0016] Preferably, in the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, after the phosphorylated modified peptides are enriched, the supernatant is collected, ACN and TFA are directly added to adjust the pH of the supernatant to 0.3-0.5, and then Ti 4+ -IMAC enrichment of glycosylated peptides.

[0017] Preferably, in the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, the biological sample is a cell sample, a lysate is added at 5-10 times the volume of the cell sample, and a crude protein extract is obtained by centrifugation; the SDS mass concentration (g / ml) in the lysate is greater than or equal to 1%.

[0018] Preferably, in the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, the biological sample is a tissue sample, a lysis solution is added according to 5-10 times the volume of the tissue sample, the protein is precipitated with methanol-chloroform, and the protein is dissolved with a buffered salt solution containing SDS to obtain a crude protein extract; the SDS mass concentration (g / ml) in the lysis solution is greater than or equal to 1%.

[0019] Preferably, in the method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, cyclodextrin or a cyclodextrin derivative is added to SDS at a molar ratio of 1 to 6:1.

[0020] In general, compared with the prior art, the above technical scheme conceived by the present invention can achieve the following beneficial effects because it is found that the SDS-cyclodextrin complex formed by adding cyclodextrin or cyclodextrin derivatives and SDS can not only avoid the influence of SDS on the enrichment of post-translationally modified peptides by antibodies, but also can use relevant antibodies to enrich post-translationally modified peptides without desalting treatment.

[0021] The method for continuously enriching multiple post-translational modifications of proteins provided by the present invention first uses a lysis solution containing SDS to lyse a biological sample to obtain a crude protein extract, then adds cyclodextrin or a cyclodextrin derivative to form an SDS-cyclodextrin complex with SDS, and then performs enzymatic hydrolysis to obtain a polypeptide mixture. Experiments have confirmed that the SDS-cyclodextrin complex can not only avoid the influence of SDS on the enrichment of post-translational modified polypeptides by antibodies, but also can directly enrich post-translational modified polypeptides such as ubiquitinated and acetylated modified polypeptides using relevant antibodies without desalting treatment, and can realize continuous enrichment of multiple different post-translational modified polypeptides in combination with relevant antibodies and other enrichment materials, and can also omit the desalting step between the enrichment of different modified polypeptides, which can reduce the loss of post-translational modified polypeptides and continuously prepare multiple different post-translational modified proteomics samples with simpler steps, shorter operation time and better repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the continuous enrichment process of four post-translationally modified polypeptides in Example 1.

[0023] Figure 2 The middle left is the mass spectrometry detection results of three groups of mammalian cells after ubiquitinated peptide enrichment, including the number of ubiquitinated peptides and the number of ubiquitination sites; the right is the quantitative correlation heat map of ubiquitination sites in the three groups of mammalian cells.

[0024] Figure 3 The middle left is the mass spectrometry detection results of three groups of mammalian cells after phosphorylated peptide enrichment, including the number of phosphorylated peptides and the number of phosphorylation sites; the right is the quantitative correlation heat map of phosphorylation sites in the three groups of mammalian cells.

[0025] Figure 4The middle left is the mass spectrometry detection results of three groups of mammalian cells after glycosylated peptide enrichment, including the number of glycosylated peptides and the number of glycosylation sites; the right is the quantitative correlation heat map of glycosylation sites in three groups of mammalian cells.

[0026] Figure 5 These are the results of mass spectrometry detection of some clinical samples after sequential enrichment, including the number of proteins, the number of ubiquitination modification sites, the number of acetylation modification sites, the number of phosphorylation modification sites, and the number of glycosylation modification sites.

[0027] Figure 6 The middle left is a scatter plot of the total number of proteins in all clinical samples before enrichment; the right is a heat map of the quantitative correlation of proteins in all clinical samples before enrichment.

[0028] Figure 7 The middle left is a scatter plot of the number of ubiquitination sites after enrichment of ubiquitinated peptides in all clinical samples; the right is a heat map of the quantitative correlation of ubiquitination sites after enrichment of ubiquitinated peptides in all clinical samples.

[0029] Figure 8 The middle left is a scatter plot of the number of acetylation sites after enrichment of acetylated modified peptides in all clinical samples; the right is a heat map of the quantitative correlation of acetylation sites after enrichment of acetylated modified peptides in all clinical samples.

[0030] Fig. 9 The middle left is a scatter plot of the number of phosphorylation sites after enrichment of phosphorylated modified peptides in all clinical samples; the right is a heat map of the quantitative correlation of phosphorylation sites after enrichment of phosphorylated modified peptides in all clinical samples.

[0031] Fig.10 The middle left is a scatter plot of the number of glycosylation sites after enrichment of glycosylated modified peptides in all clinical samples; the right is a heat map of the quantitative correlation of glycosylation sites after enrichment of glycosylated modified peptides in all clinical samples.

[0032] Fig.11 The middle left is a scatter plot of the number of phosphorylated peptides and the number of phosphorylation sites after phosphorylation-modified peptides were enriched in mouse tissue samples; the right is a heat map of the correlation of phosphorylation sites after phosphorylation-modified peptides were enriched in mouse tissue samples.

[0033] Fig.12 The middle left is a scatter plot of the number of glycosylated peptides and the number of glycosylation sites after enrichment of glycosylated modified peptides in mouse tissue samples; the right is a heat map of the correlation of glycosylation sites after enrichment of glycosylated modified peptides in mouse tissue samples.

[0034] Fig.13The middle left is a scatter plot of the number of phosphorylated peptides and the number of phosphorylation sites after the Arabidopsis leaf tissue sample was enriched with phosphorylated modified peptides; the right is a heat map of the correlation of phosphorylation sites after the Arabidopsis leaf tissue sample was enriched with phosphorylated modified peptides.

[0035] Fig.14 The middle left is a scatter plot of the number of phosphorylated peptides and the number of phosphorylation sites after the Arabidopsis leaf tissue sample was enriched with glycosylated peptides; the right is a heat map of the correlation of phosphorylation sites after the Arabidopsis leaf tissue sample was enriched with glycosylated peptides. DETAILED DESCRIPTION

[0036] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0037] The detection of protein post-translational modification generally includes three steps, namely, biological sample lysis to extract crude protein and enzymatic hydrolysis into polypeptide solution; treatment of polypeptide solution to meet enrichment conditions; elution of modified polypeptide from enriched material, and mass spectrometry detection after desalting. Among them, the enrichment of target post-translational modified polypeptide is the key. In the previous patent CN 114459848 B, a universal proteomics sample preparation method, we found that the biological sample was lysed by using SDS-containing lysis solution, and the SDS-cyclodextrin complex formed by cyclodextrin or cyclodextrin derivative treatment did not affect protein hydrolysis, and the affinity of the SDS-cyclodextrin complex with peptides such as phosphorylated peptides was quite different. The SDB-RPS filler can well separate the SDS-cyclodextrin complex and peptides. After desalting of the SDB-RPS filler, the SDB-RPS filler with adsorbed target peptides is obtained, and finally the target peptides are eluted. However, this method requires desalting to remove the SDS-cyclodextrin complex to achieve the purpose of purifying and enriching peptides, and is preferably suitable for adsorption and enrichment of phosphorylated peptides in different biological samples.

[0038] The commonly used lysis buffers for biological sample lysis mainly include three categories, namely, lysis buffers containing sodium dodecyl sulfate (SDS), lysis buffers containing urea, and lysis buffers containing sodium deoxycholate (SDC). However, when using lysis buffers containing urea, since urea is difficult to be compatible with the enrichment material, the peptide solution needs to be desalted before enriching various modified peptides. Compared with direct enrichment, enrichment after desalting takes 2-3 hours longer; and when using lysis buffers containing SDS or SDC, since SDS or SDC is difficult to be compatible with antibodies, desalting is still required when enriching various modified peptides in combination with antibodies. Compared with direct enrichment, enrichment after desalting takes longer time. Each additional desalting and vacuum drying process will result in a loss of at least 10% of the peptides. Enrichment after desalting can easily lead to a large loss of target peptides, which is a technical problem that needs to be solved urgently. Especially for continuous enrichment of proteomics samples with multiple post-translational modifications, different samples need to be desalted before enrichment, which takes a long time, has low detection efficiency and causes large sample losses.

[0039] Although the above-mentioned SDS-cyclodextrin complex does not affect proteolysis, there are obvious differences in the specific functions of antibodies and enzymes. It is not clear whether the SDS-cyclodextrin complex has an effect on the antibody enrichment effect. The present invention is improved on the basis of the preparation method provided by the previous patent CN114459848 B. The results surprisingly found that the biological sample was first lysed with a lysis solution containing SDS to obtain a crude protein extract, and then cyclodextrin or a cyclodextrin derivative was added to form an SDS-cyclodextrin complex with SDS for enzymatic hydrolysis and enrichment. It can not only avoid the influence of SDS on the enrichment of post-translationally modified peptides by antibodies, but also can directly enrich post-translationally modified peptides such as ubiquitinated and acetylated modified peptides without desalting treatment during the enrichment process. It is only necessary to desalt and remove the complex before the target modified peptide segment enters the mass spectrometry detection after antibody enrichment, which can greatly shorten the pre-treatment time and improve the detection efficiency.

[0040] Moreover, there is no need for desalting between the enrichment of different modified peptides, and the combination of relevant antibodies and other enrichment materials can achieve rapid and continuous enrichment of a variety of post-translationally modified peptides. In particular, the combination of antibody enrichment materials and metal oxide enrichment materials can achieve rapid and high-throughput continuous enrichment of a variety of protein post-translationally modified omics samples.

[0041] The present invention is improved on the basis of the preparation method in patent CN 114459848 B. The biological sample is lysed by using a lysis solution containing SDS to obtain a crude protein extract, and then cyclodextrin or a cyclodextrin derivative is added to form an SDS-cyclodextrin complex, and then pancreatic enzyme is added to enzymatically hydrolyze the protein to obtain a polypeptide solution. Antibodies are first used to enrich post-translationally modified polypeptides such as ubiquitinated and acetylated modified peptides; acetonitrile (ACN) and trifluoroacetic acid (TFA) are then used to adjust the pH of the supernatant after antibody enrichment, and phosphorylated modified peptides are enriched by enrichment materials such as metal oxides; and then acetonitrile (ACN) and trifluoroacetic acid (TFA) are used to adjust the phosphorylated polypeptide. The pH of the supernatant after enrichment, the glycosylated modified peptides are enriched by the enrichment material, and desalting and re-dissolving are not required when continuously enriching peptides of different modified types. The continuous enrichment of various post-translationally modified peptides can be performed by adjusting the pH of the solution to be enriched by acetonitrile (ACN) and trifluoroacetic acid (TFA). Since the SDS-cyclodextrin complex in the solution has no effect on the enzymatic hydrolysis and antibody enrichment of modified peptides, and there is no need to desalt and remove the complex during the continuous enrichment process, the desalting step can be omitted, and the complex only needs to be desalted and removed after the target peptide is enriched and before entering the mass spectrometry detection. In addition, this method can also simplify the adjustment of the organic phase and the acidity of the solution, which can further shorten the pre-treatment time and significantly improve the detection efficiency.

[0042] Based on this, the present invention provides a method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, which comprises the following steps:

[0043] (1) Protein extraction: Add a lysis buffer containing 0.5% to 10% SDS mass concentration (g / ml) to the biological sample to fully release and denature the protein in the sample, centrifuge or methanol-chloroform precipitation, and dissolve the protein in a buffered saline solution containing SDS to obtain a crude protein extract.

[0044] If the biological sample is a cell sample, centrifugation is used to obtain a crude protein extract; if the biological sample is a tissue sample, methanol-chloroform is used to precipitate the protein, and the protein is dissolved in a buffered salt solution containing SDS to obtain a crude protein extract.

[0045] (2) Proteolysis: Add cyclodextrin or cyclodextrin derivative solution to the crude protein extract obtained in (1), mix well to form an SDS-cyclodextrin complex, and then perform enzymatic hydrolysis to obtain a polypeptide solution-1, wherein the modified peptides in the polypeptide solution-1 include ubiquitinated modified peptides, acetylated modified peptides, phosphorylated modified peptides, and glycosylated modified peptides; preferably, the cyclodextrin or cyclodextrin derivative is added to SDS in a molar ratio of 1 to 6:1.

[0046] In some embodiments, pancreatic enzyme is added at a ratio of pancreatic enzyme to extracted sample protein of 1:(50-100), and enzymatic hydrolysis is performed to obtain polypeptide solution-1.

[0047] (3) Continuous enrichment of multiple protein post-translational modifications: first, use post-translational modification antibodies to enrich ubiquitinated and / or acetylated modified peptides, centrifuge, wash, and elute to obtain ubiquitinated modified peptide enrichment solution and / or acetylated modified peptide enrichment solution;

[0048] Acetonitrile (ACN) and trifluoroacetic acid (TFA) are then directly added to the supernatant after the above antibody enrichment to adjust the pH of the supernatant, and the enrichment material is used to enrich the phosphorylated modified peptides and / or glycosylated modified peptides.

[0049] The enrichment material for enriching phosphorylated modified peptides is IMAC, and acetonitrile (ACN) and trifluoroacetic acid (TFA) are added to adjust the pH of the supernatant to 3-4. In some embodiments, the phosphorylated modified peptide enrichment material is Fe 3+ -IMAC, add an equal volume of 1% TFA / ACN to the supernatant after antibody enrichment (i.e., add TFA to ACN and mix so that the volume proportion of TFA is 1%), adjust the volume concentration of TFA in the supernatant to 0.5%, and the pH of the solution to 3.5;

[0050] Or the enrichment material for enriching phosphorylated modified peptides is a metal oxide such as TiO2 or CaTiO3, acetonitrile (ACN) and trifluoroacetic acid (TFA) are added to adjust the pH of the supernatant to 0.05-0.15, and the present invention experiments found that TFA concentration ≥ 6% can inhibit the adsorption of non-phosphorylated polypeptides, and when SDS is used as a lysate component, the strong acidity of the supernatant TFA final concentration of 6% can be suitable for TiO2 and CaTiO3 to enrich phosphorylated polypeptides. In some embodiments, an equal volume of 12% TFA / ACN is added to the supernatant after antibody enrichment, and the TFA volume concentration in the supernatant is adjusted to 6%, and the solution pH is 0.095.

[0051] The peptide segments of phosphorylated peptides enriched by these two enrichment materials are slightly different. In the present invention, IMAC-CaTiO3 can also be used to enrich phosphorylated peptides in series, and the phosphorylated peptide segments enriched twice are mixed and then detected. In some embodiments, an equal volume of 1% TFA / ACN is first added to the supernatant after antibody enrichment, and Fe 3+ -IMAC was used to enrich phosphorylated peptides, and the supernatant was collected by centrifugation. ACN and TFA were added to the supernatant to adjust its pH to 0.8-0.1. CaTiO3 was used to enrich phosphorylated peptides again, and the eluents enriched twice were collected.

[0052] After the phosphorylated modified peptides or glycosylated modified peptides are enriched with the enrichment material, the phosphorylated modified peptide enrichment solution or glycosylated modified peptide enrichment solution is obtained through centrifugation, washing and elution.

[0053] More preferably, the phosphorylated modified peptides are first enriched with enrichment materials, and then the glycosylated modified peptides are enriched. After the phosphorylated modified peptides are enriched, the supernatant is collected, and ACN and TFA are directly added to adjust the pH of the supernatant to 0.3-0.5, and Ti 4+ -IMAC enrichment of glycosylated modified peptides. In some embodiments, the supernatant after the phosphorylated modified peptides were enriched was taken, ACN and TFA were added, and the volume concentration of TFA in the supernatant was adjusted to 3%, the volume concentration of ACN was 80%, and the pH of the solution was 0.4.

[0054] Furthermore, the lysate further comprises 5-20 mM of a reducing agent and 20-80 mM of an alkylating agent; the reducing agent is TCEP and / or DTT, and the alkylating agent is CAA and / or IAA.

[0055] The SDS-containing buffered saline solution includes a buffered saline solution containing SDS with a mass concentration (g / ml) of 1% and 100 mM Tris.

[0056] The method can also simplify the adjustment of the organic phase and the acidity of the solution, further shorten the pre-treatment time for detecting a variety of modified peptides, and improve the detection efficiency.

[0057] The following are examples

[0058] The descriptions of the reagents used in the following examples are as follows:

[0059] Adjustment solution F1: 1% TFA / ACN, which means that TFA is added to ACN so that the volume ratio of TFA is 1%. For example, 1 μl of TFA is added to ACN to a total volume of 100 μl, which is 1% TFA / ACN.

[0060] Adjustment solution F2: 12% TFA / ACN, which means that TFA is added to ACN so that the volume ratio of TFA is 12%. For example, 12 μl of TFA is added to ACN to a total volume of 100 μl, which is 12% TFA / ACN.

[0061] Enrichment material-1: Post-translational modification antibodies fixed on solid supports such as magnetic beads or agarose resin, including but not limited to ubiquitination modification, acetylation modification, methylation modification, specific types of phosphorylation modification and other modification residue motif antibodies; Enrichment material-2: IMAC beads after washing and balancing, including Fe 3+ -IMAC; Enrichment material-3: cleaned and balanced metal oxides, including but not limited to TiO2, CaTiO3; Enrichment material-4: cleaned and balanced IMAC beads, including but not limited to Ti 4+ -IMAC.

[0062] Example 1 Continuous Enrichment of Multiple Post-translationally Modified Peptides

[0063] In this example, the continuous enrichment process of multiple post-translationally modified polypeptides is as follows: Figure 1 As shown, the polypeptide solution sample is prepared based on the proteomics sample preparation method in patent CN114459848 B, specifically as follows:

[0064] 1. Protein Lysis

[0065] 1.1 For cell samples: add an appropriate amount of lysis buffer A (1% SDS, 100mM Tris-HCl, 10mM TCEP, 40mM CAA, pH 8.5), heat at 95°C for 10min, ultrasonically disrupt cells, and measure protein concentration. In this example, the SDS concentration in the lysis buffer is the mass concentration, in g / ml.

[0066] 1.2 For tissue samples: add appropriate amount of lysis buffer A (1% SDS, 100mM Tris-HCl, 10mM TCEP, 40mM CAA, pH 8.5), heat at 95℃ for 10min, fully homogenize, centrifuge at 12000rpm for 3min, take the supernatant to a new tube and use methanol-chloroform to precipitate the protein, then add appropriate amount of lysis buffer B (1% SDS, 100mM Tris-HCl, pH 8.5), sonicate to dissolve the protein precipitate, and measure the protein concentration.

[0067] 2. Preparation of Peptide Solution

[0068] Take 2 mg of protein and add solution C (250 mM HP-β-CD) at a volume ratio of 10:3. Use a pipette to blow and aspirate to mix the solution. Then add pancreatic enzyme at a protein to pancreatic enzyme mass ratio of 50:1. Perform enzymatic hydrolysis overnight at 37°C to obtain peptide solution-1. After enriching the target peptide, desalt it and then perform mass spectrometry detection.

[0069] 3. Continuous enrichment of protein post-translational modifications

[0070] 3-1 Antibody-based enrichment of post-translationally modified peptides

[0071] Centrifuge the peptide solution-1 at 12000rpm for 3min, and take the supernatant to a new tube. Add the enriched material-1 (ubiquitinated antibody agarose beads) to the supernatant, and shake at 1500rpm and 4℃ for 1 hour. Centrifuge at 3000g and 4℃ for 2min, and take the supernatant to a new tube to obtain peptide solution-2, which will be used as a tandem enrichment sample for subsequent post-translational modifications. Add 1mL of washing solution D (0.01% CHAPS / PBS) to the precipitate, centrifuge at 3000g and 4℃ for 2min to remove the supernatant, wash 3 times in total, and add 50μL of elution solution E (0.2% TFA) to elute the peptide, which is the enriched ubiquitinated modified peptide solution.

[0072] The above-mentioned enriched ubiquitinated peptide solution was desalted, and then the data were collected using U3000 liquid chromatography coupled with timsTOF Pro mass spectrometry, and the data were analyzed using FragPipe software. The results are shown in Figure 2 , more than 10,000 ubiquitination sites can be stably identified in each group of cells, and the correlation coefficient of each sample can reach above 0.7.

[0073] After the ubiquitinated protein is hydrolyzed by trypsin, a glycine-glycine group is left on the lysine residue. In the present invention, the enrichment antibody used for the ubiquitinated and acetylated peptides is not limited, including but not limited to the antibodies in this embodiment. The ubiquitinated peptides are enriched using antibodies against this structure. UbiquitinRemnant Motif (K-ε-GG) Kit, similar to ubiquitination, uses anti-acetyl group antibodies to enrich acetylated peptides, such as Acetyl-Lysine Motif [Ac-K] Kit. In the present invention, the enrichment of post-translationally modified peptides based on antibodies includes but is not limited to the enrichment of ubiquitinated and acetylated modified peptides, and can also be replaced with other antibodies to enrich methylated modified, specific types of phosphorylated modified, glutarylated modified and other peptides.

[0074] 3-2 Selective enrichment of phosphorylated peptides

[0075] ①IMAC enrichment

[0076] The supernatant after enrichment of antibody-based post-translational modified peptides does not need to be drained, desalted and re-dissolved, and can be directly used for the next enrichment. Add an equal volume of adjustment solution F1 (1% TFA / ACN) to the peptide solution-2 to make the final TFA concentration in the solution 0.5% by volume, mix thoroughly, centrifuge at 12000rpm for 3min, and take the supernatant (abbreviated as 0.5% TFA / 50% ACN) to a new tube. Add 50μL of enrichment material-2 (Fe 2+) to the supernatant at a mass: volume ratio of 8:1. 3+ -IMAC), 1500rpm, shake for 10min. After centrifugation at 2000g for 30s, rotate the EP tube 180°, centrifuge at 2000g for 30s, take the supernatant to a new tube, and obtain peptide solution-3 and enriched material-2 precipitation. Add 500μL of cleaning solution G (1% AA / 50% ACN) to the enriched material-2 precipitation tube and wash 5 times. Add 100μL of eluent J (40% ACN / 10% NH3·H2O) to elute the peptide, and collect the eluent as the enriched phosphorylated modified peptide solution 1.

[0077] ②Metal oxide enrichment

[0078] Add an equal volume of adjustment solution F2 (12% TFA / ACN) to the peptide solution-2, so that the final volume concentration of TFA in the solution is 6%, and add enrichment material-3 (TiO2 or CaTiO3) according to the mass ratio of 1:100, and shake at 1500rpm and 25℃ for 10min. After centrifugation at 2000g for 30s, rotate the EP tube 180°, centrifuge at 2000g for 30s, take the supernatant to a new tube, and obtain the peptide solution-4 and enrichment material-3 precipitation. The supernatant is used as a tandem enrichment sample for subsequent post-translational modification. Add 1mL of cleaning solution H (6% TFA / 50% ACN) to the TiO2 or CaTiO3 tube and wash 5 times. Then add 500μL of cleaning solution I (50% ACN / 0.1% TFA) to wash once. Add 100 μL of eluent J (40% ACN / 10% NH 3 · H 2 O) to elute the polypeptide, and collect the eluate to obtain the enriched phosphorylated modified polypeptide solution.

[0079] ③Tandem-Fe-IMAC-CaTiO3 tandem enrichment

[0080] IMAC enrichment: Same as above, collect eluate 1;

[0081] CaTiO3 enrichment: Add an appropriate amount of TFA to the supernatant after IMAC enrichment to adjust the final TFA concentration in the solution to 6%, add enrichment material-3 (CaTiO3) at a mass ratio of 1:100, and shake at 1500rpm and 25℃ for 10min. After centrifugation at 2000g for 30s, rotate the EP tube 180°, centrifuge at 2000g for 30s, take the supernatant to a new tube, and obtain polypeptide solution-4 and enrichment material-3 precipitation. The supernatant is used as a tandem enrichment sample for subsequent post-translational modification. Add 1mL of cleaning solution H (6% TFA / 50% ACN) to the CaTiO3 tube and wash 5 times. Then add 500μL of cleaning solution I (50% ACN / 0.1% TFA) to wash once. 100 μL of eluent J (40% ACN / 10% NH 3 · H 2 O) was added to elute the peptide, and eluent 2 was collected. The eluent 1 and the eluent 2 were mixed to obtain an enriched phosphorylated-modified peptide solution.

[0082] The above three enrichment methods can be selected at will. The enriched peptide solution is desalted, and then the data is collected using U3000 liquid chromatography in series with timsTOF Pro mass spectrometry. The data is analyzed using FragPipe software. The results are shown in Figure 3 .

[0083] Depend on Figure 3 It can be seen that more than 10,000 phosphorylation sites can be stably identified in each group of cells, and the correlation coefficient of each sample can reach more than 0.7.

[0084] 3-3 Selective enrichment of glycosylated peptides

[0085] The supernatant after phosphorylated peptide enrichment does not need to be drained, desalted and re-dissolved, and can be directly used for the next enrichment step. Peptide solution-4 is used to enrich glycosylated peptides, as follows:

[0086] Take the supernatant after phosphorylated peptide enrichment to a new tube, add appropriate amount of TFA and ACN, make the final volume concentration of TFA in the solution to be 3%, the final volume concentration of ACN to be 80%, add enrichment material-4 (TiO2) at a mass ratio of 1:10 4+ -IMAC), incubate at 25°C and 1500rpm for 30min. Then centrifuge at 2000g for 30s to remove the supernatant. Then wash the precipitate three times with 1mL of cleaning solution K (3% TFA / 80% ACN). Add 100μL of eluent L (0.1% TFA / 60% ACN) to the precipitate to elute the polypeptide, centrifuge at 12000rpm for 3min, take the supernatant to a new tube, repeat the process, and combine the supernatants. After draining the eluent, resuspend the drained polypeptide with enzymatic buffer M (50mM NH4HCO3), add 1μL PNGase F (N-glycosidase F), and digest overnight at 37°C (use PNGase F to hydrolyze asparagine-linked oligosaccharides on the polypeptide, followed by mass spectrometry detection, and identify the peptide segment based on the mass deviation). After desalting, use U3000 liquid chromatography tandem with timsTOF Pro mass spectrometry to collect data, and use FragPipe software to analyze the data. The results are as follows Figure 4 shown.

[0087] like Figure 4 It can be seen that more than 1500-2000 glycosylation sites can be stably identified in each group of cells, and the correlation coefficient of each sample can reach more than 0.7.

[0088] Example 2 Ubiquitination-acetylation-phosphorylation-glycosylation tandem enrichment for clinical samples

[0089] According to the following steps, 32 clinical tissue samples were processed respectively. The overall overview is as follows: Figure 5 .

[0090] (1) Preparation of peptide solution

[0091] According to the volume ratio of 1:(5-10), add SDS-containing lysis buffer A (1% SDS, 100mM Tris-HCl, 10mM TCEP, 40mM CAA, pH 8.5) to the tissue sample, heat it at 95℃ for 10min, homogenize it with a tissue homogenizer, centrifuge it at 12000rpm for 3min, take the supernatant into a new tube, and precipitate the protein with methanol-chloroform as follows:

[0092] Take 100μL of the supernatant solution, add 400μL of methanol, shake for 5 seconds; then add 100μL of chloroform, shake for 5s; then add 300μL of H2O, shake for 5s. 14000g, 1min. Remove the upper aqueous phase, then remove the lower organic phase, leaving only the middle protein layer, add 400μL of methanol, 21000g, 2min, remove the supernatant, add lysis buffer B (1% SDS, 100mM Tris-HCl, pH 8.5), sonicate to dissolve the protein precipitate, take a small amount of sample, and determine the protein concentration using the BCA kit.

[0093] Take 1000 μg of the protein obtained above (the required amount of protein is taken according to the concentration quantitatively determined by BCA), add solution C (250 mM HP-β-CD), and mix the solution by pipetting. Add pancreatic enzyme at a ratio of 1:50 for protein, and perform enzymatic hydrolysis at 37°C overnight. After the enzymatic hydrolysis is completed, centrifuge at 12,000 rpm for 3 minutes, take the supernatant to a new tube, and obtain polypeptide solution-1.

[0094] A small amount of peptide solution-1 was taken for proteomics detection. The experimental results are as follows Figure 6 shown.

[0095] (2) Enrichment of ubiquitinated peptides

[0096] About 30ug of enrichment material-1 (agarose beads cross-linked with ubiquitinated antibodies) was added to the above-obtained polypeptide solution-1 to enrich the target peptide segment, and the supernatant and enrichment material-1 enrichment precipitate were obtained by centrifugation. The supernatant was taken to a new tube and used as a tandem enrichment sample for subsequent post-translational modification. The enrichment material-1 was resuspended with 100mM Tris-HCl. The enrichment material-1 was added to the polypeptide solution and incubated for 1 hour at 1500rpm and 4°C on an oscillator. Subsequently, the supernatant (polypeptide solution-2) was transferred to a new EP tube after centrifugation at 3000g and 4°C for 2min.

[0097] Add 1mL of washing solution D (0.01% CHAPS / PBS) to the enriched material-1 after incubation, and invert the EP tube 7-8 times. Then centrifuge at 3000g, 4℃ for 2min, remove the supernatant, and repeat the washing twice; add 50μL of 0.2% TFA, blow and aspirate the enriched material-1 several times, place it at room temperature for 5min, collect the supernatant into a new tube, repeat once, and combine the eluate to obtain the ubiquitinated peptide enrichment solution; the obtained eluate is desalted and then tested on a mass spectrometer. The test results are as follows Figure 7 shown.

[0098] (3) Enrichment of acetylated peptides

[0099] In this embodiment, the supernatant (peptide solution-2) obtained by centrifugation after incubation with the above ubiquitination antibody agarose beads is directly subjected to the next step of acetylated polypeptide enrichment. The enrichment method is the same as that of ubiquitination, wherein a different enrichment material-2 such as acetylated antibody agarose beads is used. The obtained eluate is desalted and then tested on a mass spectrometer. The test results are as follows: Figure 8 shown.

[0100] (4) Enrichment of phosphorylated peptides

[0101] The supernatant (peptide solution-3) obtained by centrifugation after incubation with the above-mentioned acetylated antibody agarose beads can be directly used for the next step of phosphorylated peptide enrichment without desalting and re-dissolving. Take the supernatant obtained by centrifugation after incubation with the acetylated antibody agarose beads to a new tube, add an equal volume of adjustment solution F2 (12% TFA / ACN), mix well, centrifuge at 12000rpm for 3min, take the supernatant to a new EP tube, add 100μL of enrichment material-3 (CaTiO3) to each sample, shake at 25℃, 1500rpm, and shake for 10min. 2000g, 30s, obtain the enrichment material-3 precipitate, and collect the supernatant (peptide solution-4) to a new tube for the enrichment of glycosylated peptides.

[0102] Add 1 mL of cleaning solution H (6% TFA / 50% ACN) to the enriched material-3 precipitate obtained after centrifugation, mix thoroughly, centrifuge at 2000g for 30s, discard the supernatant, and repeat 4 times. Then add 1 mL of cleaning solution I (50% ACN / 0.1% TFA), mix thoroughly, centrifuge at 12000g for 2min, and discard the supernatant. Add 100 μL of eluent J (40% ACN / 10% NH3·H2O), mix thoroughly, centrifuge at 12000g for 3min, transfer the supernatant to a new tube, repeat once, and combine the eluents to obtain the phosphorylated polypeptide enrichment solution; the obtained eluent is desalted and then tested on a mass spectrometer. The test results are as follows: Fig. 9 shown.

[0103] (5) Glycosylated peptide enrichment

[0104] The supernatant obtained by centrifugation after incubation of enriched material-3 does not need to be desalted and re-dissolved, and is directly used for the next step of glycosylated polypeptide enrichment. Adjust the polypeptide solution-4 to 80% ACN / 3% TFA in the system, take 50μL of the enriched material-4 mixture and add it to the sample, 25°C, 1500rpm, and shake for 30min. Then centrifuge at 2000g for 30s and discard the supernatant. Wash the enriched material-4 precipitate three times with 1mL of cleaning solution K (3% TFA / 80% ACN), add 100μL of eluent L (0.1% TFA / 60% ACN) to the precipitate, mix thoroughly, centrifuge at 12000rpm for 3min, take the supernatant to a new tube, repeat once, and combine the eluents to obtain the glycosylated polypeptide enrichment solution.

[0105] After the eluate was dried by freeze centrifugation, the dried glycosylated polypeptides were resuspended in 50 mM enzymatic buffer M (50 mM NH4HCO3); 1 μL PnaseF was added, and the enzymatic hydrolysis was carried out at 37°C overnight. After desalting, the mass spectrometer was used for detection. The detection results were as follows: Fig.10 shown.

[0106] The time required for continuous enrichment of each modified peptide using the above method is compared with the existing method as shown in Table 1.

[0107] Table 1 Comparison of time required for continuous enrichment of modified peptides

[0108]

[0109]

[0110] This example performs ubiquitination-acetylation-phosphorylation-glycosylation tandem enrichment on clinical tissue samples, and can efficiently process more than 30 sample volumes, indicating that this tandem enrichment method can achieve good enrichment effects in clinical applications.

[0111] Example 3 Ubiquitination-acetylation-phosphorylation-glycosylation tandem enrichment for animal tissues

[0112] (1) Preparation of peptide solution

[0113] Process the four mouse tissue samples according to the following steps:

[0114] Add SDS-containing lysis buffer A (1% SDS, 100mMTris-HCl, 10mM TCEP, 40mM CAA, pH 8.5) to the mouse tissue sample at a volume ratio of 1:(5-10), heat at 95°C for 10 minutes, homogenize with a tissue homogenizer, and centrifuge at 12000rpm for 3 minutes. Take the supernatant to a new tube and use methanol-chloroform to precipitate the protein as follows:

[0115] Take 100 μL of the supernatant solution, add 400 μL of methanol, shake for 5 seconds, then add 100 μL of chloroform, shake for 5 seconds, then add 300 μL of H2O, shake for 5 seconds, and centrifuge at 14000g for 1 min; remove the upper aqueous phase, then remove the lower organic phase, leaving only the middle protein layer, add 400 μL of methanol, 21000g, 2min, remove the supernatant, and obtain the protein sample.

[0116] Add lysis solution B (1% SDS, 100mM Tris-HCl, pH 8.5) to the above protein sample, sonicate to dissolve the protein precipitate, take a small amount of solution sample, and determine the protein concentration using a BCA kit. Take 1000μg of protein, add solution C (250mM HP-β-CD), and mix the solution by pipetting. Add pancreatic enzyme at a ratio of 1:50 for pancreatic enzyme: protein, and enzymolyze at 37°C overnight. After the enzymolysis is completed, centrifuge at 12000rpm for 3min, take the supernatant to a new tube, and obtain polypeptide solution-1.

[0117] (2) Enrichment of ubiquitinated-acetylated peptides

[0118] In the present invention, polypeptide solution-1 is enriched according to the ubiquitination-modified peptide and acetylation-modified peptide enrichment method in Example 2, and the supernatant (polypeptide solution-3) obtained by centrifugation after incubation with the above-mentioned acetylation antibody agarose beads is directly used for the next step of phosphorylated polypeptide enrichment without desalting and re-dissolving.

[0119] (3) Enrichment of phosphorylated peptides

[0120] Add an equal volume of adjustment solution F2 to the supernatant (peptide solution-3), mix well, centrifuge at 12000rpm for 3min, take the supernatant to a new EP tube, add 100μL of enrichment material-3 to each sample, shake at 25℃, 1500rpm, 10min. 2000g, 30s, get the enrichment material-3 precipitate, and collect the supernatant (peptide solution-4) to a new tube for glycosylated peptide enrichment.

[0121] Add 1 mL of cleaning solution H (6% TFA / 50% ACN) to the enriched material-3 precipitate, mix thoroughly, centrifuge at 2000g for 30s, discard the supernatant, and repeat 4 times. Then add 1 mL of cleaning solution I (50% ACN / 0.1% TFA), mix thoroughly, centrifuge at 12000g for 2min, and discard the supernatant. Add 100 μL of eluent J (40% ACN / 10% NH3·H2O), mix thoroughly, centrifuge at 12000g for 3min, transfer the supernatant to a new tube, repeat once, and combine the eluents to obtain the phosphorylated polypeptide enrichment solution; the obtained eluent is desalted and tested on a mass spectrometer, and the test results are as follows: Fig.11 shown.

[0122] (4) Glycosylated peptide enrichment

[0123] The supernatant after incubation of enriched material-3 is used for the next step of glycosylated polypeptide enrichment. Adjust the polypeptide solution-4 to 80% ACN / 3% TFA in the system, take 50μL of the enriched material-4 mixture and add it to the sample, 25°C, 1500rpm, and shake for 30min. Then centrifuge at 2000g for 30s and discard the supernatant. Wash the enriched material-4 precipitate three times with 1mL of cleaning solution K (3% TFA / 80% ACN), add 100μL of eluent L (0.1% TFA / 60% ACN) to the enriched material-4 precipitate, mix thoroughly, centrifuge at 12000rpm for 3min, take the supernatant to a new tube, repeat once, and combine the eluents to obtain the glycosylated polypeptide enrichment solution.

[0124] After the eluate was dried by freezing centrifugation, the dried glycosylated peptides were resuspended in 50mM enzymatic buffer M (50mM NH4HCO3). 1μL PnaseF was added and enzymatically digested at 37℃ overnight. After desalting, the samples were tested by mass spectrometry. The test results were as follows: Fig.12 shown.

[0125] In this example, knockout mice were used as the experimental group and wild-type mice were used as the control group. The results showed that mammalian tissue materials can obtain good enrichment effects through the tandem enrichment method.

[0126] In this embodiment, selective enrichment can be performed according to the target peptides to be enriched. For example, the peptide solution-1 can be added directly with the adjustment solution F2 to enrich the phosphorylated peptides (i.e., phosphorylated peptides) according to the above method without enriching the ubiquitinated and acetylated peptides.

[0127] Example 4 Ubiquitination-acetylation-phosphorylation-glycosylation tandem enrichment for plant tissues

[0128] (1) Preparation of peptide solution

[0129] Three Arabidopsis leaf tissue samples were processed according to the following steps:

[0130] Leaf tissue samples were added with an appropriate amount of lysis buffer A (1% SDS, 100mM Tris-HCl, 10mM TCEP, 40mM CAA, pH 8.5), heated at 95°C for 10min, homogenized with a tissue homogenizer, and centrifuged at 12000rpm for 3min. The supernatant was transferred to a new tube, and the protein was precipitated with methanol-chloroform as follows:

[0131] Take 100μL of the supernatant solution, add 400μL of methanol, and shake for 5 seconds. Add 100μL of chloroform and shake for 5s. Add 300μL of H2O and shake for 5s. 14000g, 1min. Remove the upper aqueous phase, and then remove the lower organic phase, leaving only the middle protein layer. Add 400μL of methanol, 21000g, 2min. Remove the supernatant to obtain the protein sample.

[0132] Lysis solution B (1% SDS, 100 mM Tris-HCl, pH 8.5) was added to the obtained protein sample, the protein precipitate was dissolved by ultrasonication, a small amount of sample was taken, and the protein concentration was determined using a BCA kit.

[0133] Take 1000 μg protein, add solution C (250 mM HP-β-CD), and mix the solution by pipetting. Add pancreatic enzyme at a ratio of 1:50 for protein, and perform enzymatic hydrolysis at 37°C overnight. After the enzymatic hydrolysis is completed, centrifuge at 12000 rpm for 3 minutes, and take the supernatant (peptide solution-1) to a new tube.

[0134] (2) Enrichment of ubiquitinated-acetylated peptides

[0135] In the present invention, polypeptide solution-1 is enriched according to the ubiquitination-modified peptide and acetylation-modified peptide enrichment method in Example 2, and the supernatant (polypeptide solution-3) obtained by centrifugation after incubation with the above-mentioned acetylation antibody agarose beads is directly used for the next step of phosphorylated polypeptide enrichment without desalting and re-dissolving.

[0136] (3) Enrichment of phosphorylated peptides

[0137] Add an equal volume of adjustment solution F-2 to the supernatant (peptide solution-3), mix well, and centrifuge at 12000rpm for 3min. Take the supernatant to a new EP tube. Add 100μL of enrichment material-3 to each sample, shake at 25℃, 1500rpm, and shake for 10min. 2000g, 30s, to obtain the enrichment material-3 precipitate, and collect the supernatant (peptide solution-4) to a new tube.

[0138] Add 1 mL of cleaning solution H (6% TFA / 50% ACN) to the enriched material-3 precipitate, mix thoroughly, centrifuge at 2000g for 30s, discard the supernatant, and repeat 4 times. Then add 1 mL of cleaning solution I (50% ACN / 0.1% TFA), mix thoroughly, centrifuge at 12000g for 2min, and discard the supernatant. Add 100 μL of eluent J (40% ACN / 10% NH3·H2O), mix thoroughly, centrifuge at 12000g for 3min, transfer the supernatant to a new tube, repeat once, and combine the eluents to obtain the phosphorylated polypeptide enrichment solution; the obtained eluent is desalted and tested on a mass spectrometer. The test results are as follows: Fig.13 shown.

[0139] (4) Glycosylated peptide enrichment

[0140] Take the supernatant (peptide solution-4) after incubation with enrichment material-3 for the next step of glycosylated polypeptide enrichment. Adjust the polypeptide solution-4 to 80% ACN / 3% TFA, take 50μL of the enrichment material-4 mixture and add it to the sample, 25°C, 1500rpm, and shake for 30min. Then centrifuge at 2000g for 30s and discard the supernatant. Wash the enrichment material-4 precipitate three times with 1mL of cleaning solution K (3% TFA / 80% ACN). Add 100μL of eluent L (0.1% TFA / 60% ACN) to the precipitate, mix thoroughly, centrifuge at 12000rpm for 3min, take the supernatant to a new tube, repeat once, and combine the eluents to obtain the glycosylated polypeptide enrichment solution.

[0141] After freezing and centrifuging the eluate, resuspend the glycosylated peptides in 50mM enzymatic buffer M (50mM NH4HCO3). Add 1μL PnaseF, enzymatically digest at 37℃ overnight, and detect by mass spectrometry after desalting. The test results are as follows: Fig.14 shown.

[0142] Although the present embodiment does not set up an experimental group and a control group, the experiment was conducted using Arabidopsis leaf tissues with three replicates. The results show that plant tissue materials can achieve good enrichment effects through the tandem enrichment method.

[0143] In this embodiment, selective enrichment can be performed according to the target peptides to be enriched. For example, the peptide solution-1 can be added directly with the adjustment solution F2 to enrich the phosphorylated peptides (i.e., phosphorylated peptides) according to the above method without enriching the ubiquitinated and acetylated peptides.

[0144] In the present invention, the enrichment materials, cleaning solutions, eluents, and enzymatic hydrolysis buffers used in the above embodiments are not limited. All existing enrichment materials, cleaning solutions, eluents, and enzymatic hydrolysis buffers used to enrich the relevant target modified peptide segments are included in the protection scope of the present invention.

[0145] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications, characterized in that: The following steps are involved: (1) Protein extraction: Add lysis buffer containing SDS at a concentration of 0.5% to 10% g / mL to the biological sample to obtain a crude protein extract; (2) Proteolysis: add cyclodextrin or cyclodextrin derivative solution to the crude protein extract obtained in (1) and mix well to form SDS-cyclodextrin complex, followed by pancreatic enzymatic hydrolysis to obtain polypeptide solution; (3) Continuously enriching multiple post-translational modifications of proteins: first, using a post-translational modification antibody to enrich the ubiquitinated and / or acetylated modified peptides in the polypeptide solution in (2), centrifuging, washing, and eluting to obtain a ubiquitinated modified polypeptide enrichment solution and / or an acetylated modified polypeptide enrichment solution; ACN and TFA are then directly added to the supernatant after antibody enrichment to adjust the pH of the supernatant, and the phosphorylated modified peptides and / or glycosylated modified peptides are enriched using the enrichment material; The phosphorylation-modified peptides are first enriched using enrichment materials, and then the glycosylation-modified peptides are enriched; The enrichment material for enriching phosphorylated modified peptides is IMAC, and the pH of the supernatant after antibody enrichment is adjusted to 3-4, and / or the enrichment material for enriching phosphorylated modified peptides is a metal oxide, and the pH of the supernatant after antibody enrichment is adjusted to 0.05-0.15, and the metal oxide is TiO2 or CaTiO3; After enrichment of phosphorylated peptides, the supernatant was collected and ACN and TFA were added directly to adjust the pH of the supernatant to 0.3-0.

5. 4+ -IMAC enrichment of glycosylated peptides.

2. The method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications according to claim 1, characterized in that: IMAC-CaTiO3 was used to enrich phosphorylated peptides in series, and the eluates from the two enrichments were collected.

3. The method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications according to claim 2, characterized in that: First, add an equal volume of 1% TFA / ACN to the supernatant after antibody enrichment, and then use Fe 3+ -IMAC was used to enrich phosphorylated peptides, and the supernatant was collected by centrifugation. ACN and TFA were added to the supernatant to adjust its pH to 0.8-0.

1. CaTiO3 was used to enrich phosphorylated peptides again, and the eluents enriched twice were collected.

4. The method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications according to claim 3, characterized in that: The biological sample is a cell sample, and a lysis solution is added according to 5-10 times the volume of the cell sample, and centrifugation is performed to obtain a crude protein extract; the SDS mass concentration in the lysis solution is greater than or equal to 1% g / mL.

5. The method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications according to claim 3, characterized in that: The biological sample is a tissue sample, and a lysis solution is added according to 5-10 times the volume of the tissue sample, the protein is precipitated with methanol-chloroform, and the protein is dissolved with a buffered salt solution containing SDS to obtain a crude protein extract; the mass concentration of SDS in the lysis solution is greater than or equal to 1% g / mL.

6. The method for rapid high-throughput continuous enrichment of multiple protein post-translational modifications according to claim 4 or 5, characterized in that: The cyclodextrin or cyclodextrin derivative is added to SDS at a molar ratio of 1 to 6:1.

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