Hydrophilic two-dimensional nano composite material as well as preparation and application thereof
By enriching phosphorylated and glycosylated peptides under neutral conditions using hydrophilic two-dimensional nanocomposites MOLs@PEI@Au@L-Cys, the problem of difficulty in simultaneously enriching N-phosphorylated peptides in the prior art is solved, and high coverage and selectivity analysis is achieved, and biocompatibility is enhanced.
Patent Information
- Application Number
- CN202311240281.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-25
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art is difficult to simultaneously enrich the phosphorylated and glycosylated peptides, especially N-phosphorylated peptides under neutral conditions, and there are problems of signal interference and low abundance limitation.
The hydrophilic two-dimensional nanocomposite MOLs@PEI@Au@L-Cys were used to achieve enrichment and analysis under neutral conditions by using high organic phase as loading buffer solution and aqueous phase as elution buffer solution.
The simultaneous enrichment of phosphorylated and glycosylated peptides under neutral conditions, especially N-phosphorylated peptides, improve the coverage and selectivity of the analysis, reduce non-specific adsorption interference, and enhance biocompatibility.
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Abstract
Description
Technical Field
[0001] The present invention relates to the preparation and application of a hydrophilic two-dimensional nanocomposite material, which has the characteristics of high coverage, strong selectivity, excellent hydrophilicity, good biocompatibility, etc., and can simultaneously enrich phosphorylated peptides, especially N-phosphorylated peptides and glycosylated peptides under neutral conditions. Through this technology, the information of cell phosphorylation and glycosylated peptides can be stably obtained, so as to explore their dynamic changes, which provides a prerequisite for further exploring the biological functions of post-translationally modified proteins. Background Art
[0002] As two of the most typical and widespread post-translational modifications (PTMs), protein glycosylation and phosphorylation show important roles in early non-invasive diagnosis and targeted therapy of diseases. Abnormal glycosylated and phosphorylated peptides are often used in clinical proteomics research and disease-related biomarkers (Anal. Chem., 2020, 92, 3, 2680–2689). Compared with O-phosphorylated peptides and N-linked glycopeptides, which can also be stably enriched under acidic conditions, phosphorylation on His, Arg or Lys residues, i.e., N-phosphorylation, involves a (PN) bond formed by the -NH group and the γ-phosphoramidite of ATP. This PN bond has a high hydrolysis ΔG° (-12 to -14 kcal / mol) and is unstable in hot alkali and acidic environments (Int. J. Biol. Macromol., 2020, 145, 53-63), so the study of N-phosphorylation remains challenging. At present, the combination of bottom-up proteomics research and high-sensitivity mass spectrometry (MS) is considered to be an important tool for the comprehensive analysis of phosphorylated and glycosylated proteins. After the protein is digested into peptides, it is analyzed by mass spectrometry (MS) to create sequence tags for database searches (Front Chem 2022, 10, 1091243). However, for most biological samples, it is difficult to directly identify post-translationally modified proteins by MS. Because the inherent obstacles of low abundance and poor ionization efficiency of phosphorylated and glycosylated proteins / peptides limit their high-sensitivity analysis. In addition, coexisting substances such as unmodified peptides, surfactants, salts and other impurities are complex, resulting in strong interference with the target molecule signal during MS analysis. Therefore, the separation and enrichment process of proteins / peptides before MS is essential.
[0003] At present, some methods have been developed for the separate enrichment of glycopeptides and phosphorylation, including boric acid affinity chromatography, hydrazine chemistry, hydrophilic interaction chromatography, lectin affinity chromatography and metabolic labeling (J.Sep.Sci, 202200292) for N-linked glycopeptide enrichment, while immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography, immunoprecipitation and ion exchange chromatography are commonly used for phosphopeptide enrichment (Trac-Trend.Anal.Chem, 2016, 78, 70-83). However, due to the acid instability and structural multiplicity of N-pho, it is difficult to induce immunity by classical methods, and the current O-phosphorylated peptide enrichment method under acidic conditions is not suitable for the enrichment of N-phosphorylated peptides (Nat.Commun, 2020, 11, 6226). Hydrophilic interaction chromatography (HILIC) is of interest for its simple operation, excellent reproducibility, satisfactory compatibility with MS, and unbiased affinity for glycopeptides / phosphopeptides (Trac-Trend. Anal. Chem, 2023, 165, 117148). Hydrophilic materials are considered to be the key to HILIC, as they can interact specifically with the hydroxyl groups on the glycoside chains and the phosphate groups of phosphopeptides. Therefore, hydrophilic materials are an ideal means for simultaneously enriching phosphorylated and glycosylated peptides, especially for effectively capturing N-phosphorylated peptides under neutral conditions.
[0004] Two-dimensional metal organic sheets (MOLs) are a new type of two-dimensional tunable functional material. They not only inherit the advantages of MOF's uniform structure, ultra-high specific surface area and sustainable pores, but also provide more accessible active sites (Ange, 201512054), which makes it more hydrophilic and more conducive to further functionalization. The MOLs used in this study were synthesized by solvent thermal synthesis from ZrCl4 and 1,3,5-tri(4-carboxyphenyl)benzene. Polyethyleneimine (PEI) is a water-soluble polymer. Due to its high density of amino groups, it is easy to post-functionalize and has excellent hydrophilicity. It is used as a reducing agent and stabilizer to load gold nanoparticles on the surface of metal organic sheet materials (MOLs). Then, zwitterionic L-cysteine is fixed through Au-S bonds, which can enhance the hydrophilicity of the material while enhancing stability and further reducing nonspecific adsorption. The synthesized MOLs@PEI@Au@L-Cys can achieve the simultaneous enrichment of phosphorylated and glycosylated peptides, especially N-phosphorylated peptides, under neutral conditions. Finally, in order to enhance sequence coverage and protein identification, upstream hydrophilic enrichment was combined with downstream high pH RPLC fractionation to achieve universal enrichment and analysis of phosphorylated and glycosylated peptides from whole proteins of prokaryotic cells to eukaryotic cells, and the regulation of phosphorylated and glycosylated modified proteins on eukaryotic cell membranes was studied. Summary of the invention
[0005] The present invention utilizes the specific hydrophilic interaction with the hydroxyl group on the glycoside chain and the phosphate group of the phosphopeptide to develop a method based on hydrophilic interaction chromatography for simultaneously enriching phosphorylated and glycosylated peptides including N-phosphorylation under neutral conditions.
[0006] To achieve the above object, the technical solution adopted by the present invention is:
[0007] (1) Design and preparation of hydrophilic two-dimensional nanocomposite materials MOLs@PEI@Au@L-Cys. This material uses polyethyleneimine (PEI) as a reducing agent and stabilizer to load gold nanoparticles on the surface of metal organic sheets (MOLs), and then fixes L-cysteine through Au-S bonds to achieve hydrophilic modification of the two-dimensional metal organic sheets (MOLs).
[0008] (2) Using a high organic phase as a loading buffer solution: ≥75% (v / v) acetonitrile, and an aqueous phase as an elution buffer solution to achieve hydrophilic enrichment and elution, and adding ammonium bicarbonate solution to the loading buffer solution and the elution buffer solution to ensure a neutral environment, so that the pH value of the loading buffer and the elution buffer is maintained at 7-7.5.
[0009] (3) The phosphorylated and glycosylated peptides in the cell enzymatic peptide fragments are enriched by the hydrophilic two-dimensional nanocomposite in the loading buffer and eluted in the elution buffer solution.
[0010] (4) Combine mass spectrometry analysis to achieve deep coverage of proteome qualitative and quantitative analysis, with applications in brain science, synthetic biology, and precision medicine.
[0011] The cells include one or more of tumor cells, immune cells, stem cells, healthy cells, bacteria, fungi, and microorganisms.
[0012] The technical solution adopted by this method is as follows:
[0013] 1. Synthesis and characterization of hydrophilic two-dimensional nanocomposites
[0014] 1.1 Synthesis
[0015] According to the following synthetic route (Attached Figure 1 ) to obtain a hydrophilic two-dimensional nanocomposite material. Polyethyleneimine (PEI) was used as a reducing agent and stabilizer to load gold nanoparticles on the surface of metal organic sheets (MOLs), and then L-cysteine was fixed through Au-S bonds to achieve hydrophilic modification of the two-dimensional metal organic sheets (MOLs).
[0016] The synthesis process of the hydrophilic two-dimensional nanocomposite MOLs@PEI@Au@L-Cys includes:
[0017] (1) MOLs were fully dispersed in deionized water, ultrasonically dispersed, and a polyethyleneimine (PEI) solution with a concentration of 10-100 mg / mL was added, with the ratio of MOLs to PEI being 1:20-30 (m / m). The mixture was shaken and centrifuged to obtain a PEI-functionalized two-dimensional nanocomposite (MOLs@PEI).
[0018] (2) MOLs@PEI was added to deionized water to form a dispersion, and a tetrachloroauric acid solution with a concentration of 10-100 mg / mL was added, and the mass ratio of MOLs@PEI to tetrachloroauric acid was 1:1-2 (m / m). The mixture was reacted in a 70°C water bath for 1-2 h, and after centrifugation, a two-dimensional nanocomposite material functionalized with nanogold (MOLs@PEI@Au) was prepared;
[0019] (3) Deionized water was added to the prepared MOLs@PEI@Au to form a dispersion, and L-cysteine was added. The mass ratio of MOLs@PEI@Au to L-cysteine was 1:60-121. The reaction was carried out at room temperature for 24-48 hours to prepare an L-cysteine-functionalized two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys.
[0020] 1.2 Characterization
[0021] Fourier infrared spectroscopy and x-ray photoelectron spectroscopy were used to determine the synthesis of the material, a thermogravimetric analyzer was used to determine the amount of L-cysteine grafted, and a contact angle meter was used to determine the contact angle of the material. Scanning electron microscopy and transmission electron microscopy were used to display the morphology of the material.
[0022] 2. Validation of the selectivity and stability of enrichment
[0023] 2.1 Enrichment selectivity
[0024] Under certain interference, the synthesized hydrophilic two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys was used to simultaneously enrich the phosphorylated and glycosylated labeled peptides under certain interference, and the enriched peptides were analyzed by MALDI-TOF mass spectrometry.
[0025] 2.2 Enrichment stability
[0026] The synthesized hydrophilic two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys was used for three biological replicate enrichments, that is, the complete proteolysis products of HeLa cells cultured in three dishes at the same time were enriched separately, and the enrichment results were verified by Pearson correlation coefficient.
[0027] 3. Enrichment of phosphorylated and glycosylated peptides in prokaryotic and mammalian cells
[0028] The synthesized hydrophilic two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys was used to enrich the whole proteolytic peptides of E.Coli cells and HeLa cells, respectively, and high pH RPLC fractionation was combined to enhance the sequence coverage and protein identification. Finally, the enrichment results were analyzed by Orbitrap Fusion Lumos mass spectrometer.
[0029] 4. Enrichment of phosphorylated and glycosylated peptides in mammalian cell membrane systems
[0030] The synthesized hydrophilic two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys was used to enrich the proteolytic peptides of the HeLa cell membrane system, and high pH RPLC fractionation was combined to enhance the sequence coverage and protein identification. Finally, the enrichment results were analyzed by Orbitrap Fusion Lumos mass spectrometer.
[0031] This enrichment strategy based on hydrophilic two-dimensional nanocomposites has the following innovations and advantages: (1) High coverage, capable of simultaneously enriching phosphorylated and glycosylated peptides, especially N-phosphorylated peptides, under neutral conditions; (2) Good selectivity, the interaction between metal ions and hydrophilicity can effectively reduce the interference of nonspecific adsorption and improve enrichment selectivity; (3) The hydrophilicity and biocompatibility of the material are good, and the hydrophilic two-dimensional nanocomposites have the advantages of multiple hydrophilic groups and multiple metal ions, which give them good hydrophilicity and biocompatibility; (4) The hydrophilic two-dimensional nanocomposites have a large density and can be precipitated by low-speed centrifugation, which can achieve rapid separation of target peptides from non-target peptides; (5) This strategy is combined with mass spectrometry analysis for the identification and analysis of phosphorylated and glycosylated peptides in different biological samples in cell and tissue samples, providing a powerful tool for proteomics analysis and the discovery of therapeutic targets. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 .Synthesis route of hydrophilic two-dimensional nanocomposite MOLs@PEI@Au@L-Cys.
[0033] Figure 2.Characterization diagram of the MOLs@PEI@Au@L-Cys material prepared in Example 1; wherein, (a) is the fingerprint of the MOLs@PEI@Au@L-Cys material, (b) is the X-ray photoelectron spectrum of the MOLs@PEI@Au@L-Cys material, (c) and (d) are the contact angles of MOLs@PEI@Au before and after L-cysteine grafting, (e) is the amount of L-Cys grafted by thermogravimetric analysis of the MOLs@PEI@Au@L-Cys material, (f) is the nitrogen adsorption-desorption curve of the MOLs@PEI@Au@L-Cys material; (gl) is the scanning electron microscope and mapping analysis diagram of the MOLs@PEI@Au@L-Cys material.
[0034] Figure 3 .MALDI-TOF MS analysis of hydrophilic enriched phosphorylated and glycosylated labeled peptides by MOLs@PEI@Au@L-Cys.
[0035] Figure 4 .Stability of MOLs@PEI@Au@L-Cys enrichment—Pearson correlation coefficient.
[0036] Figure 5 Example 3 Phosphorylation and glycosylation sites identified in Escherichia coli and the number of corresponding proteins.
[0037] Figure 6 In Example 4, WebLogo performed sequence analysis on the phosphorylation and glycosylation sites identified in HeLa lysate.
[0038] Figure 7 .GO analysis results of membrane proteins with both phosphorylation and glycosylation modifications in HeLa cells. DETAILED DESCRIPTION
[0039] The following is a detailed description of the implementation methods of the present invention in conjunction with the attached tables and drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
[0040] Example 1
[0041] Synthesis and characterization of hydrophilic two-dimensional nanocomposite materials
[0042] 1.1 Synthesis
[0043] (1) MOLs synthesis
[0044] Take 14.0 mg (0.06 mmol) of ZrCl4 and 12.5 mg (0.0285 mmol) of 1,3,5-tris(4-carboxyphenyl)benzene into bolted tube A, and add 0.3 mL of ultrapure water and 0.88 mL of formic acid (HCOOH) into A. Ultrasonicate for 3 min, seal, and place in a 120°C thermostat for 48 h to obtain MOLs.
[0045] (2) Synthesis of MOLs@PEI@Au@L-Cys (see attached Figure 1 )
[0046] Weigh 1 mg of MOLs, add 1 mL of deionized water, and sonicate for 0.5 h to obtain a 1 mg / mL MOLs dispersion. Add 0.3 mL of 100 mg / mL PEI solution to the dispersion, mix thoroughly, and shake vigorously for 1 h. Centrifuge at 16000 g for 10 min to remove unreacted PEI, and prepare a PEI-functionalized two-dimensional nanocomposite material named MOLs@PEI.
[0047] 1 mL of deionized water was added to the prepared MOLs@PEI, and the mixture was shaken vigorously to form a MOLs@PEI dispersion; 20 μL of 100 mg / mL HAuCl4·H2O solution was added, and the mixture was mixed thoroughly and reacted in a 70°C water bath for 1 h. The free nanogold was removed by centrifugation at 16000 g to prepare a nanogold functionalized two-dimensional nanocomposite material, named MOLs@PEI@Au. 2 mL of deionized water was added to the prepared MOLs@PEI@Au, and then 121 mg of L-cysteine was added. The mixture was reacted for 24 h, and the supernatant was removed by centrifugation to prepare a L-cysteine functionalized two-dimensional nanocomposite material, named MOLs@PEI@Au@L-Cys.
[0048] 1.2 Characterization
[0049] The synthesized MOLs@PEI@Au@L-Cys composite material has the same fingerprint as L-Cys (e.g. Figure 2 a), prove the successful synthesis of materials, X-ray photoelectron spectroscopy (XPS) (such as Figure 2 b) characterization also proves this point. The contact angles of the material before and after grafting with L-cysteine were 47.1° and 20.5°, respectively, proving that the material exhibits good hydrophilicity after grafting with L-cysteine (e.g. Figure 2 cd). Thermogravimetric analysis determined that the grafting amount of L-Cys was 60.48%. Figure 2 e). Determination of nitrogen adsorption-desorption curves shows that the material has a porous structure (such as Figure 2f). Scanning electron microscopy and mapping analysis of the material showed that the synthesized material exhibited a two-dimensional structure and gold, zirconium, sulfur and other elements were evenly distributed on the surface of the material (such as Figure 2 gl).
[0050] Example 2
[0051] Verification of selectivity and stability of MOLs@PEI@Au@L-Cys enrichment
[0052] 2.1 Selectivity of Enrichment
[0053] (1) Enzymatic hydrolysis of standard protein samples
[0054] The protein samples were bovine β-casein and human immunoglobulin. 1 mg was taken and dissolved in 1 mL of 50 mM NH4HCO3 solution containing 8 M urea. A final concentration of 25 mM dithiothreitol (DTT) was added. The mixture was reacted in a 37°C water bath for 1 h. After cooling to room temperature, a final concentration of 50 mM iodoacetamide (IAA) was added. The mixture was reacted at 37°C in the dark for 30 min. Then 50 mM NH4HCO3 was added to dilute the urea concentration to 1 mg / mL. Trypsin was added to the pretreated protein solution at a ratio of 50 / 1 (w / w) of protein:trypsin, and the mixture was reacted in a 37°C water bath for 12 h. The protein enzymatic hydrolyzate was desalted with a commercial SPE trapping column and freeze-dried for later use.
[0055] (2) MOLs@PEI@Au@L-Cys hydrophilic enrichment of phosphorylated and glycosylated labeled peptides
[0056] The MOLs@PEI@Au@L-Cys hydrophilic two-dimensional nanocomposite material was dispersed in a loading buffer (75% ACN (v / v) 20 mM NH4HCO3 PH = 7.5) to prepare a 10 mg / mL dispersion, and the standard protein hydrolysate was also dissolved in the above loading buffer to prepare a 1 mg / mL solution. Take 10 μL of the composite material dispersion, add 60 μL of loading buffer, then add 10 μg of commercial N-phosphorylated standard peptide (TSpHYSIMAR) and 10 μg of bovine β-casein hydrolysate and 10 μg of human immunoglobulin G hydrolysate at the same time, mix thoroughly and shake at 4°C for 15 min, then centrifuge at 16000 rpm to remove the supernatant; elute the non-target peptides three times with 30 μL of loading buffer, and finally add 30 μL of elution buffer (20 mM NH4HCO3 PH = 7.5) and shake for 15 min to elute the target peptide twice, combine the eluates and add 0.3 μL of N-glycosidase F (25000 units / mg protein) to continue enzymatic hydrolysis for 12 h, dry and redissolve in 2% ACN and analyze by MALDI-TOF MS. After enrichment, the phosphorylated and glycosylated peptides are specifically captured (the results after enrichment are shown in Figure 2). Figure 3 ).
[0057] Unless otherwise specified, all ratios in the text are volume ratios, the loading buffer is (75% ACN (v / v) 20 mM NH4HCO3 PH=7.5), and the elution buffer is (20 mM NH4HCO3 PH=7.5).
[0058] 2.2 Stability of enrichment
[0059] (1) Extraction and enzymatic hydrolysis of whole protein from HeLa cells
[0060] Three dishes of HeLa cells were cultured under the same conditions. HeLa cells were cultured in a humidified incubator with 5% CO2 and 95% air at 37°C in DMEM medium containing 10% FBS and 1% PBS. All cells that reached 80% confluence were separated with 0.25% trypsin / ethylenediaminetetraacetic acid (EDTA) and centrifuged (500×g, 5min) to collect the cells. The cells were washed three times with cold PBS and resuspended in lysis buffer (8M urea, 10mM PBS, 1% (v / v) protease inhibitor cocktail and 1% (v / v) phosphatase inhibitor cocktail). After ultrasonic treatment in an ice bath for 120s (20s intervals every 10s), the insoluble part was separated from the soluble part by centrifugation at 16000X g for 30min at 4°C, and the supernatant was taken and the protein concentration was determined using the BCA assay. 1M DTT was added to a final concentration of 25mM and reacted in a 37°C water bath for 1h. After cooling to room temperature, 500mM IAA was added to a final concentration of 50mM, and the mixture was reacted in the dark at 37°C for 30min. 1M DTT was added to a final concentration of 50mM to quench the excess IAA. The protein was precipitated with methanol / chloroform, re-dissolved with 20mM HEPES buffer, and trypsin (protein: trypsin = 1:30, m / m) was added and incubated at 37°C for 13 hours, and then freeze-dried for use. Three cell proteolysis products were obtained.
[0061] (2) Enrichment of phosphorylated and glycosylated peptides in HeLa cells
[0062] The enrichment process of HeLa cell hydrolysate was the same as that in 2.1(2). 0.75 mg of each of the three cell hydrolysate was dissolved in 0.2 mL of loading buffer to form a dispersion. The two-dimensional nanocomposite was also dispersed in the loading buffer to prepare a 10 mg / mL dispersion. 0.3 mL of the hydrophilic two-dimensional nanocomposite was mixed with the HeLa cell hydrolysate dispersion and shaken at 4°C for 15 min. The supernatant was then removed by centrifugation at 16,000 rpm. Non-target peptides were eluted three times with 0.2 mL of loading buffer. Finally, 0.2 mL of elution buffer (20 mM NH4HCO3) was added and shaken for 15 min to elute the target peptide twice. The eluates were combined and 2 μL of N-glycosidase F (25,000 units / mg protein) was added to continue enzymatic hydrolysis for 12 h. After drying, it was redissolved in 2% ACN and analyzed by Orbitrap Fusion Lumos to obtain three biological replicates. All the data collected by mass spectrometry were searched using MaxQuant_1.6.3.3 with built-in Andromeda search engine, and the standardless quantitative mode was set. After data processing using Perseus1.6.15.0 software, the Pearson correlation coefficients were all greater than 0.8, proving that the enrichment stability of the material was good (such as the attached Figure 4 ).
[0063] Example 3
[0064] Enrichment of phosphorylated and glycosylated peptides in Escherichia coli by MOLs@PEI@Au@L-Cys
[0065] (1) Extraction and enzymatic hydrolysis of whole proteins from prokaryotic cells
[0066] E. coli (strain K12) was cultured overnight at 37°C in Luria–Bertani medium (LB, 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L tryptone) and harvested by centrifugation at 4000 × g for 5 min. The cells were washed three times with cold PBS and resuspended in 1 mL of lysis buffer (8 M urea, 10 mM PBS, 1% (v / v) protease inhibitor cocktail, and 1% (v / v) phosphatase inhibitor cocktail). After sonication in an ice bath for 180 s (20 s intervals every 10 s), the insoluble fraction was separated from the soluble fraction by centrifugation at 16000 × g for 30 min at 4°C, and the supernatant was taken and the protein concentration was determined using the BCA assay. 1 M DTT was added to a final concentration of 25 mM and reacted in a 37°C water bath for 1 h. After cooling to room temperature, 500 mM IAA was added to a final concentration of 50 mM, and the mixture was reacted at 37°C in the dark for 30 min. 1 M DTT was added to a final concentration of 50 mM to quench the excess IAA. The protein was precipitated by the methanol / chloroform method, and after re-dissolving in 20 mM HEPES buffer, the resulting protein was added with trypsin (protein: trypsin = 1:30) and incubated at 37°C for 12 hours before freeze-drying for later use.
[0067] (2) Enrichment of phosphorylated and glycosylated peptides in Escherichia coli cells
[0068] 7 mg of Escherichia coli cell protease was dissolved in 0.6 mL of loading buffer to form a dispersion. The two-dimensional nanocomposite material was also dispersed in the loading buffer to prepare a 10 mg / mL dispersion. 0.9 mL of hydrophilic two-dimensional nanocomposite dispersion was added to the cell enzymatic product dispersion and mixed thoroughly, then shaken at 4°C for 15 min, then centrifuged at 16000 rpm to remove the supernatant, 0.4 mL of loading buffer was used to elute the non-target peptides three times, and finally 0.3 mL of elution buffer (20 mM NH4HCO3) was added and shaken for 15 min to elute the target peptide twice. The eluates were combined and 5 μL of N-glycosidase F (25000 units / mg protein) was added to continue enzymatic hydrolysis for 12 h to obtain an enzymatic solution containing the target peptide.
[0069] (3) High pH classification
[0070] Agilent 1290 HPLC with self-made high pH C18 column 5 μm Durashell C18 silica particles (2.1 mm id × 150 mm) were used for target peptide separation. The mobile phase was prepared and adjusted to pH 10.0 with 28% NH3·H2O as follows. Buffer A: 2% (v / v) ACN with 10 mM ammonium acetate, buffer B: 80% (v / v) ACN with 10 mM ammonium acetate. At a flow rate of 0.3 mL / min, an 80 min separation gradient (70 min from 0 to B, 5 min from 45 to 100% B, and 5 min at 100% B) was applied. Peptides were collected from each minute elution time window from 10 to 69 min, respectively (window 1: 10 min, window 2: 11 min, ..., window 60: 69 min). For E. coli, 60 equally spaced windows were combined into 10 fractions.
[0071] (4) NanoRPLC-ESI-MS / MS
[0072] Each fraction of the enriched phosphorylated and glycosylated peptides was automatically loaded onto a RP capture column (150 μm i.d. × 5 cm) and separated by a C18 capillary column. Both the capture column and the analytical column were filled with 5 μm, Venusil XBP C18 silica particles. Two mobile phases A: 0.1% (v / v) FA aqueous solution and B: 80% ACN (0.1% FA, v / v) produced a 121-minute gradient with a flow rate of 600 nL / min (90 minutes from 7% to B, 20 minutes from 25% to 40% B, 1 minute from 40% to 80% B, and 10 minutes kept at 80% B). An Orbitrap Fusion Lumos mass spectrometer (Thermo-Fisher, San Jose, CA, USA) was operated in full scan (120,000 FWHM, 350-1500 m / z) mode in positive ion mode with an orbital trap detector. The electrospray voltage was 3.5 kV and the heated capillary temperature was 320°C. All mass spectra were recorded with Xcalibur software (version 3.1, Thermo Fisher Scientific, USA). MS / MS spectra were collected in data-dependent acquisition mode, and the total cycle time was set to 3 s. Peptides with charge states ≥ 2 were selected for sequencing and fragmented using HCD (collision energy 32%, maximum injection time 35 ms) and neutral loss trigger (△ 97.98) EThcD (ETD reaction time 50 ms, maximum ETD reagent injection time 200 ms, supplementary activation energy 25%, maximum injection time 35 ms). All product ions were detected in the ion trap.
[0073] (5) Data analysis
[0074] All mass spectrometry data were searched using MaxQuant_1.6.5.0 with built-in Andromeda search engine. After classification, 796 phosphorylation sites were identified in E. coli, corresponding to 524 proteins, including 420 N-phosphorylation sites. In addition, 128 glycosylation sites were identified in E. coli, corresponding to 119 proteins (results are shown in Figure 5 ).
[0075] Example 4
[0076] Enrichment of phosphorylated and glycosylated peptides in HeLa cells by MOLs@PEI@Au@L-Cys
[0077] The processes of HeLa cell culture, protein enzymatic hydrolysis and enrichment are the same as those in Example 2. The high pH fractionation and NanoRPLC-ESI-MS / MS analysis are the same as steps (3-5) of Example 3, except that 60 equally spaced windows are merged into 20 fractions. 7165 phosphorylation sites were identified in the HeLa cell enzymatic hydrolysate, including 3018 N-phosphorylation sites and 4147 O-phosphorylation sites, corresponding to 1719 and 2094 proteins, respectively. In addition, 1135 glycosylation sites were identified in HeLa cells, corresponding to 672 proteins. WebLogo was used to perform sequence analysis on the phosphorylation and glycosylation sites identified in the HeLa lysate. The number of serine and proline around the N-phosphorylation and O-phosphorylation sites is relatively large, while the number of lysine and serine around the N-glycosylation sites is relatively large (such as Figure 6 ).
[0078] Example 5
[0079] Enrichment of phosphorylated and glycosylated peptides in HeLa cells
[0080] After culturing HeLa cells, membrane proteins were extracted using the MinuteTM kit. The enzymatic hydrolysis and enrichment process of membrane proteins was the same as in Example 2, and the high pH fractionation and NanoRPLC-ESI-MS / MS analysis process were the same as steps (3-5) of Example 3, except that 60 equally spaced windows were merged into 6 fractions. 408 N-phosphorylation sites and 565 O-phosphorylation sites were identified in HeLa cells, corresponding to 280 and 348 proteins, respectively. In addition, 1796 glycosylation sites were identified, corresponding to 748 proteins. GO analysis of 62 proteins with both phosphorylation and glycosylation modifications revealed that the proteins were distributed in 22 membrane components including the cell membrane and nuclear membrane, and participated in biological processes including cell adhesion, magnesium ion homeostasis, and regulation of cell migration (results as shown in Figure 7 shown).
Claims
1. A method for preparing a hydrophilic two-dimensional nanocomposite material, characterized in that: The hydrophilic two-dimensional nanocomposite material is MOLs@PEI@Au@L-Cys, which is a sheet material synthesized by using metal organic sheet material (MOLs) as a base material, polyethyleneimine (PEI) as a reducing agent and stabilizer, loading gold nanoparticles (AuNPs) on the surface of MOLs, and then fixing L-cysteine through Au-S bonds.
2. The preparation method according to claim 1, characterized in that: The synthesis process of the hydrophilic two-dimensional nanocomposite MOLs@PEI@Au@L-Cys includes: (1) MOLs were fully dispersed in deionized water, ultrasonically dispersed, and polyethyleneimine (PEI) solution was added at a ratio of 1:20-30 (m / m), shaken for reaction, and centrifuged to obtain PEI-functionalized two-dimensional nanocomposites (MOLs@PEI); (2) MOLs@PEI was added to deionized water to form a dispersion, tetrachloroauric acid was added at a ratio of 1:1-2 (m / m) and reacted in a water bath at 70°C for 1-2 h. After centrifugation, a two-dimensional nanocomposite material functionalized with nanogold (MOLs@PEI@Au) was prepared; (3) Deionized water was added to the prepared MOLs@PEI@Au to form a dispersion, and L-cysteine was added. The mass ratio of MOLs@PEI@Au to L-cysteine was 1:60-121. The reaction was carried out at room temperature for 24-48 hours to prepare an L-cysteine-functionalized two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys.
3. The preparation method according to claim 2, characterized in that: The concentration of the polyethyleneimine (PEI) solution in step (1) is 10-100 mg / mL; The concentration of the tetrachloroauric acid solution in step (2) is 10-100 mg / mL.
4. A hydrophilic two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys prepared according to any one of claims 1 to 3, characterized in that: MOLs@PEI@Au@L-Cys presents a porous two-dimensional sheet structure, whose surface contains zirconium ions and gold ions, as well as hydrophilic groups such as carboxyl and hydroxyl groups.
5. An application of the hydrophilic two-dimensional nanocomposite material MOLs@PEI@Au@L-Cys as claimed in claim 4 for simultaneously enriching phosphorylated peptides and glycosylated peptides.
6. The use according to claim 5, characterized in that: Under neutral conditions (pH = 7-7.5), the cell enzymatic solution and MOLs@PEI@Au@L-Cys were fully mixed in the loading buffer, incubated at 4°C with vigorous shaking for 10-15 min, centrifuged to remove the supernatant, added the loading buffer to wash the non-target peptides, and finally added the elution buffer to shake for 10-15 min to elute the phosphorylated and glycosylated peptides, and centrifuged to remove the precipitate; The supernatant was fractionated by high pH RPLC at pH = 10 and then analyzed by mass spectrometry.
7. The use according to claim 6, characterized in that: The mass ratio of cell enzymatic peptide fragments to MOLs@PEI@Au@L-Cys in the loading buffer is 1 to 5:1 (preferably 2-3:1, more preferably 2.5:1, m / m).
8. The use according to claim 6, characterized in that: The neutral condition means that the pH values of the loading buffer and the elution buffer are both 7-7.5, the loading buffer is 75%-80% acetonitrile (v / v) 20mM ammonium bicarbonate, and the elution buffer is 5-20mM ammonium bicarbonate.