CN111808164A - Phosphorylated protein enrichment method based on multi-hydrogen bond responsive polymer - Google Patents
Phosphorylated protein enrichment method based on multi-hydrogen bond responsive polymer Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
Abstract
The present invention relates to the fields of analytical chemistry and post-translational modification proteomics. The invention provides a method for enriching and separating phosphorylated protein, which takes a multi-hydrogen bond responsive polymer as an enrichment material and adopts a column solid phase extraction mode or a dispersed solid phase extraction mode to extract phosphorylated protein from a complex biological sample. The multi-hydrogen bond responsive polymer material is obtained by copolymerizing isopropyl acrylamide and thiourea derivatives on the surface of a substrate material by a surface initiation-atom transfer radical polymerization method. The method realizes the high-selectivity enrichment of the phosphorylated protein by optimizing the enrichment parameters such as the types and concentrations of organic solvents, buffer salt types and concentrations, pH, temperature and the like of the loading solution, the eluent and the eluent. The method has the advantages of high selectivity, large adsorption capacity, flexible and controllable method, simple and easy operation and the like, and is suitable for high-selectivity enrichment of phosphorylated proteins in biological samples.
Description
Technical Field
The invention relates to the field of analytical chemistry and post-translational modification proteomics, in particular to an enrichment method of phosphorylated protein.
Background
Protein phosphorylation is a specific and reversible protein posttranslational modification, and participates in and regulates various physiological processes such as signal transduction, gene expression, cell proliferation, differentiation, apoptosis and the like in a human body. A number of studies have shown that abnormal expression of protein phosphorylation is closely related to a number of critical diseases (cancers C.E.Antai, A.M.Hudson, E.Kang, C.Zanca, C.Wirth, N.L.Stephenson, E.W.Trotter, L.L.Gallegos, C.J.Miller, F.B.Fumari, T.Hunter, J.Brognard and A.C.Newton, Cell,2015,160, 489-fold 502; J.M.Drake, E.O.Paull, N.A.Graham, J.K.Lee, B.A.Smith, B.Titz, T.Stoyanova, C.M.Farmeier, V.Unnalog, D.E.Carlin, D.T.T.K.M.C.K.K.K.K.K.K.K.K.K.K.K.K.K.C.C.C.C.D.C.M.F.F.F.F.F.F.C.C.C.F.C.H.C.H.C.C.C.H.C.H.C.H.C.C.C.C.C.H.H.C.C.C.H.C.C.C.H.H.C.H.C.C.C.H.C.C.D.H.C.H.C.C.C.C.C.D.H.C.C.D.D.C.C.H.H.C.C.D.D.D.D.H.H.C.C.C.C.H.C.D.C.C.H.H.D.D.D.D.H.H.H.C.C.C.D.H.H.C.D.H.C.C.C.H.C.C.C.C.H.C.C.C.H.H.D.D.C.C.D.H.H.H.C.C.H.D.C.H.D.C.H.H.C.H.H.H.H.H.H.H.C.C.C.C.C.C.H.C.C.C.C.C.C.H.H.C.H.H.H.H.H.C.H.H.H.H.D.C.C.H.C.C.H.H.C.C.D.C.D.D.D.D.D.D.D.D. Phosphorylated proteins are therefore of increasing scientific interest in the fields of biology, pathology and therapeutics. Due to the characteristics of low phosphorylated protein content, low stoichiometry, easy degradation and the like, the research on phosphorylated proteomes is more difficult than the research on conventional proteomics, and the phosphorylated protein enrichment is very necessary before mass spectrometry. However, due to the complex structure of phosphorylated proteins (W.Yan, J.ZHou, M.Sun, J.Chen, G.Hu and B.Shen, amino acids,2014,46, 1419-. Currently, co-immunoprecipitation is the most commonly used method for the enrichment of phosphorylated proteins. However, due to the specificity of the antibody-antigen reaction, the enrichment efficiency and selectivity of this method is limited by the antibody of choice. Depending on the phosphorylation residues, many commercially available antibodies are only effective for tyrosine phosphorylated proteins, while the vast majority (99%) of serine and threonine phosphorylated proteins are difficult to enrich due to their small epitopes and large steric hindrance (Y.Bian, L.Li, M.Dong, X.Liu, T.Kaneko, K.Cheng, H.Liu, C.Voss, X.Cao, Y.Wang, D.Litchfield, M.Ye, S.S.C.Li and H.Zou, Nat.Chem.biol.,2016,12, 959-. In addition, the expensive price of antibodies also limits their widespread use in phosphoproteomics research. The development of new materials and new methods for high-throughput, high-selectivity and high-sensitivity phosphorylated protein enrichment is an urgent need at present.
The invention discloses a phosphorylated protein enrichment method based on a multi-hydrogen bond responsive polymer, which is characterized in that isopropyl acrylamide and thiourea derivatives are copolymerized on the surface of a substrate material to obtain the material by utilizing a surface initiation-atom transfer radical polymerization reaction mechanism, and the high-selectivity enrichment of phosphorylated protein in a complex biological sample is realized by optimizing enrichment parameters such as the type and concentration of an organic solvent, the type and concentration of buffer salt, pH, temperature and the like by adopting a column Solid Phase Extraction (SPE) mode or a dispersed solid phase extraction (dSPE) mode.
Disclosure of Invention
The invention aims to provide a method for enriching phosphorylated protein from a complex sample, which has the advantages of high selectivity, high flux, wide coverage rate, strong universality and simple operation. The method uses a responsive polymer material with multiple hydrogen bonds, adopts a column Solid Phase Extraction (SPE) mode or a dispersed solid phase extraction (dSPE) mode, and realizes the high-selectivity enrichment of phosphorylated proteins in a complex biological sample by optimizing enrichment parameters such as the type and concentration of an organic solvent, the type and concentration of buffer salt, pH, temperature and the like.
The invention is realized by adopting the following technical scheme:
the multi-hydrogen bond responsive polymer is a bi-component copolymer, and the bi-component copolymer has a molecular structure shown as follows:
wherein x is 0.1-0.9; n-102～106(ii) a R is amino acid, monosaccharide, disaccharide, dipeptide, oligopeptide or zwitterionic compound with the number of amino acids of 2-4; matrix is a base material.
In the above-mentioned scheme, the first step of the method,the substrate is made of Si, Cu, Ag, Au, Pt, CuO or Fe3O4Porous SiO2Porous Al2O3Porous TiO 22Or porous ZrO2Any one or a mixture of two or more of them in any proportion; the particle diameter of the substrate material is 0.2-50 μm, and the pore diameter is
In the above scheme, the multiple hydrogen bond responsive polymer material is obtained by copolymerizing isopropyl acrylamide and thiourea derivatives on the surface of a substrate material by using a surface initiation-atom transfer radical polymerization reaction mechanism, and the specific synthetic steps are disclosed in patent 201611019827.2.
In the above scheme, the column solid phase extraction mode (SPE) specifically comprises the following steps:
firstly, loading a multi-hydrogen bond responsive polymer material as a phosphorylated protein enrichment material on a pipette tip with a sieve plate at the tail end or an SPE (solid phase extraction) small column, flushing the enrichment material by using eluent, balancing the enrichment material by using sample liquid, and then loading a biological sample dissolved in the sample liquid on the enrichment material, wherein the proportional relation between the biological sample and the sample liquid is that 0.01-1 mu g of the biological sample is dissolved in every 1 mu L of the sample liquid. Then adopting eluent to wash the enrichment material to remove non-phosphorylated protein, and finally eluting phosphorylated protein on the enrichment material by using eluent to obtain phosphorylated protein;
the volume of the eluent used for washing the enrichment material is 2-50 times of the volume of the enrichment material, the volume of the sample liquid used for balancing the enrichment material is 2-50 times of the volume of the enrichment material, the volume of the sample liquid containing the biological sample is 2-100 times of the volume of the enrichment material, the volume of the eluent used for eluting the enrichment material is 2-100 times of the volume of the enrichment material, and the volume of the eluent used for eluting the phosphorylated protein is 2-30 times of the volume of the enrichment material.
In the above scheme, the dispersed solid phase extraction mode (dSPE) specifically comprises the following steps:
placing the multi-hydrogen bond responsive polymer material as a phosphorylated protein enrichment material in a centrifuge tube, flushing the enrichment material by using eluent, balancing the enrichment material by using sample liquid, mixing the enrichment material with the sample liquid dissolved with a biological sample for incubation, wherein the proportion relation between the biological sample and the sample liquid is that 0.01-1 mu g of the biological sample is dissolved in each 1 mu L of the sample liquid. And (2) incubating the sample and the enrichment material for 2-30 minutes at the incubation temperature of 15-50 ℃, centrifuging and discarding supernatant after incubation is finished, leaching the rest precipitate with leacheate and oscillating, centrifuging and discarding supernatant after oscillation, eluting the rest precipitate with eluent, oscillating, centrifuging and taking supernatant to obtain phosphorylated protein.
The oscillation speed is 100-2500rpm, and the oscillation time is 2-30 minutes.
The volume of the eluent used for washing the enrichment material is 3-500 times of the volume of the enrichment material, the volume of the sample liquid used for balancing the enrichment material is 3-500 times of the volume of the enrichment material, the volume of the sample liquid containing the biological sample is 3-2000 times of the volume of the enrichment material, the volume of the eluent used for eluting the enrichment material is 3-3000 times of the volume of the enrichment material, and the volume of the eluent used for eluting the phosphorylated protein is 5-200 times of the volume of the enrichment material.
In the column Solid Phase Extraction (SPE) mode or the dispersed solid phase extraction (dSPE) mode:
the sample loading solution is a mixed solution of a buffer solution consisting of acid and buffer salt and an organic solvent, the volume ratio of the organic solvent is 0-50%, the mass concentration of the acid is 0-5%, the concentration of the buffer salt is 0-200mM, and the pH value of the sample loading solution is within the range of 1-7;
the leacheate consists of a mixed solution of a buffer solution consisting of acid and buffer salt and an organic solvent, wherein the volume ratio of the organic solvent is 0-50%, the mass concentration of the acid is 0-5%, the concentration of the buffer salt is 0-200mM, and the pH value of the leacheate is within the range of 1-7;
the eluent is a mixed solution of a buffer solution consisting of acid and buffer salt and an organic solvent, the volume ratio of the organic solvent is 0-50%, the mass concentration of the acid is 0-5%, the concentration of the buffer salt is 0-200mM, and the pH value of the eluent is within the range of 7-12.
The organic solvent is one or more of acetonitrile, methanol and ethanol; the buffer salt is one of ammonium formate, ammonium acetate, ammonium bicarbonate, Tris (hydroxymethyl) aminomethane (Tris) and phosphate buffer salt solution; the acid is one or more of formic acid, acetic acid and trifluoroacetic acid.
The invention has the following advantages:
1. the phosphorylated protein enrichment material prepared by the invention has the characteristics of high selectivity, high flux and the like when phosphorylated protein is separated and enriched.
2. The phosphorylated protein enrichment material prepared by the invention has flexible and adjustable solvent responsiveness, flexible method and high sensitivity, and is different from the conventional enrichment material.
3. The phosphorylated protein enrichment material prepared by the invention can be conveniently filled into extraction columns with different lengths and different inner diameters, can be directly added into a centrifugal tube, and is simple to operate and easy to repeat.
4. The phosphorylated protein obtained by enrichment can be directly used for electrospray-mass spectrometry (ESI-MS) or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the detection limit and sensitivity of the mass spectrometry are improved.
Drawings
FIG. 1 is a mass spectrum diagram of phosphorylated proteins in bovine serum albumin BSA and phosphorylated protein beta-casein (mass ratio 10:1) enriched by using a multi-hydrogen bond responsive polymer in a dSPE mode.
FIG. 2 is a mass spectrum diagram of phosphorylated proteins in bovine serum albumin BSA and phosphorylated protein beta-casein (mass ratio 50:1) enriched by using a multi-hydrogen bond responsive polymer in a dSPE mode.
FIG. 3 is a mass spectrum diagram of phosphorylated proteins in bovine serum albumin BSA and phosphorylated protein beta-casein (mass ratio 100:1) enriched by using a multi-hydrogen bond responsive polymer in a dSPE mode.
FIG. 4 is a mass spectrum diagram of phosphorylated proteins in bovine serum albumin BSA and phosphorylated protein alpha-casein (mass ratio 1000:1) enriched by using a multi-hydrogen bond responsive polymer in a dSPE mode.
FIG. 5 is a mass spectrum of phosphorylated proteins in BSA and phosphorylated protein beta-casein (mass ratio 2000:1) enriched by using a multi-hydrogen bond responsive polymer in dSPE mode.
Detailed Description
In order to make the contents, technical solutions and advantages of the present invention more apparent, the present invention is further described below with reference to specific embodiments and drawings, and these embodiments are merely used to illustrate the present invention, and the present invention is not limited to the following embodiments.
The raw materials and equipment used in the examples:
the multiple hydrogen bond responsive polymers used in the examples can be synthesized according to patent 201611019827.2. Bovine Serum Albumin (BSA), beta-casein, alpha-casein were purchased from Sigma-Aldrich (China). Acetonitrile, methanol, formic acid, acetic acid, commercially available from alpha company. Trifluoroacetic acid (TFA) was purchased from TEDIA, USA. Other reagents were used in commercial analytical purity. Mass spectrometry results were obtained by ESI-Q-TOF MS.
Example 1
And taking the multi-hydrogen bond responsive polymer as an enrichment material, and enriching the phosphorylated protein in an SPE mode. The enrichment material is loaded with 1mg of the multi-hydrogen bond responsive polymer enrichment material into tip, the enrichment material is washed by 30 mu L of 5% acetonitrile/10% ammonia water solution, the enrichment material is balanced by 50 mu L of 5% acetonitrile/acetic acid-ammonium acetate (buffer salt concentration is 5mmol/mL) buffer solution, 10 mu g of phosphorylated protein beta-casein and 10 mu g of interferent bovine serum albumin BSA are dissolved in 30 mu L of 5% acetonitrile/acetic acid-ammonium acetate (buffer salt concentration is 5mmol/mL) buffer solution and loaded on the enrichment material (hereinafter referred to as loading). After loading, the enrichment material was rinsed with 100 μ L of 5% acetonitrile/acetic acid-ammonium acetate buffer (5 mmol/mL buffer salt) to remove non-phosphorylated proteins, and finally phosphorylated proteins on the material were eluted with 30 μ L of 5% acetonitrile/10% ammonia to obtain phosphorylated proteins.
Example 2
And taking the multi-hydrogen bond responsive polymer as an enrichment material, and enriching the phosphorylated protein in an SPE mode. 1mg of the multiple hydrogen bond responsive polymer enriched material was loaded into tip, the enriched material was washed with 30. mu.L of 10mmol/mL ammonium bicarbonate solution, the enriched material was equilibrated with 50. mu.L of 1% formic acid solution, and 10. mu.g of phosphorylated protein alpha-casein and 10. mu.g of the interferent bovine serum albumin BSA were dissolved in 30. mu.L of 1% formic acid solution and loaded. After loading, the enrichment material is rinsed with 100 μ L of 1% formic acid solution to remove non-phosphorylated proteins, and finally, 30 μ L of 10mmol/mL ammonium bicarbonate solution is used to elute phosphorylated proteins on the material, thereby obtaining phosphorylated proteins.
Example 3
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. 3mg of the multi-hydrogen bond responsive polymer enrichment material is filled into a centrifuge tube, 50 mu L of 1% ammonia water solution is adopted to wash the enrichment material, then 100 mu L of 3% acetonitrile/0.1% formic acid solution is used to balance the enrichment material, 10 mu g of phosphorylated protein beta-casein and 20 mu g of interferent bovine serum albumin BSA are dissolved in 50 mu L of 3% acetonitrile/0.1% formic acid solution to be loaded, the loading and the mixing and the incubation are carried out on the enrichment material, the oscillation rotating speed of the incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining precipitate was rinsed with 120. mu.L of 3% acetonitrile/0.1% formic acid solution and shaken at 2000rpm for 5 minutes. After shaking, centrifuging to remove supernatant, eluting the rest precipitate with 30 μ L of 1% ammonia water solution, shaking at 2500rpm for 10 min, and centrifuging to obtain supernatant to obtain phosphorylated protein.
Example 4
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. The method comprises the steps of loading 5mg of the multi-hydrogen bond responsive polymer enrichment material into a centrifuge tube, flushing the enrichment material by 50 mu L of 10% ammonia water solution, then balancing the enrichment material by 100 mu L of 3% acetonitrile/formic acid-ammonium formate (buffer salt concentration is 5mmol/mL), dissolving 10 mu g of phosphorylated protein beta-casein and 100 mu g of interferent bovine serum albumin BSA in 200 mu L of 3% acetonitrile/formic acid-ammonium formate (buffer salt concentration is 5mmol/mL) buffer solution, loading the mixture, mixing the mixture with the enrichment material, and incubating, wherein the oscillation rotating speed of incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining precipitate was rinsed with 500. mu.L of 3% acetonitrile/formic acid-ammonium formate (buffer salt concentration 5mmol/mL) buffer solution and shaken at 2000rpm for 5 minutes. And after shaking, centrifuging to remove supernatant, eluting the rest precipitate with 30 μ L10% ammonia water solution, shaking at 2500rpm for 10 min, and centrifuging to obtain supernatant to obtain phosphorylated protein. The enrichment result is shown in fig. 1, the mass spectrum of the eluent only has a stronger signal of phosphorylated protein beta-casein, and does not have a signal of non-phosphorylated protein BSA, which indicates that the multi-hydrogen bond responsive polymer enrichment material successfully and selectively enriches phosphorylated protein from a complex biological sample mixture.
Example 5
And taking the multi-hydrogen bond responsive polymer as an enrichment material, and enriching the phosphorylated protein in an SPE mode. Loading 1mg of the multiple hydrogen bond responsive polymer enriched material into tip, washing the enriched material with 30. mu.L of 30% acetonitrile/1% ammonia solution, balancing the enriched material with 50. mu.L of 5% acetonitrile/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution, dissolving 10. mu.g of phosphorylated protein alpha-casein and 10. mu.g of interferent bovine serum albumin BSA in 30. mu.L of 5% acetonitrile/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution, loading, the enriched material was eluted with 60. mu.L of 5% acetonitrile/formic acid-ammonium formate (buffer salt concentration 5mmol/mL) buffer solution to remove non-phosphorylated proteins, and finally phosphorylated proteins on the material were eluted with 30. mu.L of 30% acetonitrile/1% ammonia to obtain phosphorylated proteins.
Example 6
And taking the multi-hydrogen bond responsive polymer as an enrichment material, and enriching the phosphorylated protein in an SPE mode. Loading 1.5mg of the poly-hydrogen bond-responsive polymer-enriched material into tip, washing the enriched material with 30. mu.L of 50% methanol solution, equilibrating the enriched material with 50. mu.L of 10% methanol/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution, loading 10. mu.g of phosphorylated protein beta-casein and 20. mu.g of the interferent bovine serum albumin BSA in 100. mu.L of 10% methanol/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution, washing the enriched material with 300. mu.L of 10% methanol/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution to remove non-phosphorylated proteins after loading, and finally eluting phosphorylated proteins on the material with 30. mu.L of 50% methanol solution to obtain phosphorylated proteins.
Example 7
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. 10mg of the multi-hydrogen bond responsive polymer enrichment material is loaded into a centrifuge tube, 50 mu L of 20mmol/mL ammonium bicarbonate solution is adopted to wash the enrichment material, then 100 mu L of 10% acetonitrile/0.1% trifluoroacetic acid solution is used to balance the enrichment material, 10 mu g of phosphorylated protein beta-casein and 500 mu g of interferent bovine serum albumin BSA are dissolved in 800 mu L of 10% acetonitrile/0.1% trifluoroacetic acid solution for sample loading, and the enrichment material is mixed and incubated, the oscillation speed of the incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining precipitate was rinsed with 2.5mL of a 15% acetonitrile/0.1% trifluoroacetic acid solution and shaken at 2000rpm for 5 minutes. After shaking, centrifuging to remove supernatant, eluting the rest precipitate with 30 μ L of 20mmol/mL ammonium bicarbonate solution at 2500rpm for 10 min, and centrifuging to obtain supernatant to obtain phosphorylated protein. The enrichment results are shown in FIG. 2. The mass spectrum of the eluent only has a stronger signal of phosphorylated protein beta-casein and does not have a signal of non-phosphorylated protein BSA, which indicates that the multi-hydrogen bond responsive polymer enrichment material successfully and selectively enriches phosphorylated protein from a complex biological sample mixture.
Example 8
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. The method comprises the steps of loading 5mg of the multi-hydrogen bond responsive polymer enrichment material into a centrifuge tube, flushing the enrichment material by 50 mu L of 50% methanol/1% ammonia water solution, balancing the enrichment material by 100 mu L of 30% methanol/0.1% trifluoroacetic acid solution, dissolving 10 mu g of phosphorylated protein alpha-casein and 100 mu g of interferent bovine serum albumin BSA in 500 mu L of 30% methanol/0.1% trifluoroacetic acid solution, loading the mixture, mixing the mixture with the enrichment material, and incubating at the oscillation rotation speed of 1000rpm, the incubation time of 30 minutes and the incubation temperature of 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining precipitate was rinsed with 1mL of 30% methanol/0.1% trifluoroacetic acid solution and shaken at 2000rpm for 5 minutes. After shaking, centrifuging to remove supernatant, eluting the rest precipitate with 30 μ L of 50% methanol/1% ammonia water at 2500rpm for 10 min, shaking, and centrifuging to obtain supernatant to obtain phosphorylated protein.
Example 9
And taking the multi-hydrogen bond responsive polymer as an enrichment material, and enriching the phosphorylated protein in an SPE mode. Loading 1.5mg of the poly-hydrogen bond-responsive polymer-enriched material into tip, washing the enriched material with 30. mu.L of 50% ethanol solution, balancing the enriched material with 50. mu.L of 10% ethanol/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution, loading 10. mu.g of phosphorylated protein beta-casein and 20. mu.g of the interferent bovine serum albumin BSA in 200. mu.L of 10% ethanol/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution, washing the enriched material with 500. mu.L of 10% ethanol/formic acid-ammonium formate (buffer salt concentration of 5mmol/mL) buffer solution to remove non-phosphorylated protein after loading, and finally eluting phosphorylated protein on the material with 30. mu.L of 50% ethanol solution to obtain phosphorylated protein.
Example 10
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. 10mg of the multi-hydrogen bond responsive polymer enrichment material is loaded into a centrifuge tube, 50 mu L of 50% ethanol/1% ammonia water solution is adopted to wash the enrichment material, then 100 mu L of 30% ethanol/0.1% trifluoroacetic acid solution is used to balance the enrichment material, 10 mu g of phosphorylated protein beta-casein and 1mg of interferent bovine serum albumin BSA are dissolved in 2mL of 30% ethanol/0.1% trifluoroacetic acid solution for sample loading, and the enrichment material is mixed and incubated, the oscillation speed of the incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining precipitate was rinsed with 5mL of 30% ethanol/0.1% trifluoroacetic acid solution and shaken at 2000rpm for 5 minutes. After shaking, centrifuging to remove supernatant, eluting the rest precipitate with 50 μ L50% ethanol/1% ammonia water solution at 2500rpm for 10 min, shaking, and centrifuging to obtain supernatant to obtain phosphorylated protein. The enrichment results are shown in FIG. 3. The mass spectrum of the eluent has only stronger signal of phosphorylated protein beta-casein and no signal of non-phosphorylated protein BSA, which shows that when the non-phosphorylated protein of the interferent is increased to 100 times (mass ratio) of the phosphorylated protein, the multi-hydrogen bond responsive polymer enrichment material can still be successfully and selectively enriched to the phosphorylated protein from the complex biological sample mixture.
Example 11
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. The method comprises the steps of loading 5mg of multi-hydrogen bond responsive polymer enrichment material into a centrifuge tube, flushing the enrichment material by 50 mu L of 50mmol/mL ammonium bicarbonate solution, balancing the enrichment material by 100 mu L of 5% ethanol/1% formic acid solution, dissolving 10 mu g of phosphorylated protein alpha-casein and 50 mu g of interference bovine serum albumin BSA in 500 mu L of 5% ethanol/1% formic acid solution, loading the mixture, mixing the mixture with the enrichment material, and incubating, wherein the incubation rotation speed is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining precipitate was rinsed with 800. mu.L of 5% ethanol/1% formic acid solution and shaken at 2000rpm for 5 minutes. After shaking, centrifuging to remove supernatant, eluting the rest precipitate with 50 μ L50 mmol/mL ammonium bicarbonate solution at 2500rpm for 10 min, shaking, and centrifuging to obtain supernatant to obtain phosphorylated protein.
Example 12
And taking the multi-hydrogen bond responsive polymer as an enrichment material, and enriching the phosphorylated protein in an SPE mode. Loading 1mg of multi-hydrogen bond responsive polymer enrichment material into tip, flushing the enrichment material by using 30 mu L of 20mmol/mL ammonium bicarbonate solution, balancing the enrichment material by using 50 mu L of 30% methanol solution, dissolving 10 mu g of phosphorylated protein alpha-casein and 10 mu g of interferent bovine serum albumin BSA in 30 mu L of 30% methanol solution for sample loading, eluting the enrichment material by using 100 mu L of 30% methanol solution to remove non-phosphorylated protein after sample loading, and finally eluting phosphorylated protein on the material by using 30 mu L of 20mmol/mL ammonium bicarbonate solution to obtain phosphorylated protein.
Example 13
And taking the multi-hydrogen bond responsive polymer as an enrichment material, and enriching the phosphorylated protein in an SPE mode. Loading 1mg of multi-hydrogen bond responsive polymer enrichment material into tip, flushing the enrichment material by using 30 mu L of 50% acetonitrile/3% ammonia water solution, balancing the enrichment material by using 50 mu L of 30% acetonitrile solution, dissolving 10 mu g of phosphorylated protein beta-casein and 10 mu g of interferent bovine serum albumin BSA in 30 mu L of 30% acetonitrile solution for sample loading, leaching the enrichment material by using 100 mu L of 30% acetonitrile solution for removing non-phosphorylated protein after sample loading, and finally eluting phosphorylated protein on the material by using 30 mu L of 50% acetonitrile/3% ammonia water solution to obtain phosphorylated protein.
Example 14
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. 10mg of the multi-hydrogen bond responsive polymer enrichment material is filled into a centrifuge tube, 50 mu L of 10% ammonia water solution is adopted to wash the enrichment material, then 100 mu L of 10% ethanol solution is used to balance the enrichment material, 10 mu g of phosphorylated protein alpha-casein and 10mg of interference bovine serum albumin BSA are dissolved in 10mL of 10% ethanol solution for sample loading, the mixture and the enrichment material are mixed and incubated, the oscillation speed of incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining pellet was rinsed with 10mL of 15% ethanol solution and shaken at 2000rpm for 5 minutes. And after shaking, centrifuging to remove supernatant, eluting the rest precipitate with 50 mu L of 10% ammonia water solution, shaking at 2500rpm for 10 min, and centrifuging to obtain supernatant to obtain phosphorylated protein. The enrichment results are shown in FIG. 4. The mass spectrum of the eluent has only the signal of phosphorylated protein beta-casein and no signal of non-phosphorylated protein BSA, which shows that when the non-phosphorylated protein of the interferent is increased to 1000 times (mass ratio) of the phosphorylated protein, the multi-hydrogen bond responsive polymer enrichment material can still be successfully and selectively enriched to the phosphorylated protein from the complex biological sample mixture.
Example 15
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. 10mg of the multi-hydrogen bond responsive polymer enrichment material is filled into a centrifuge tube, 50 mu L of 10% ammonia water solution is adopted to wash the enrichment material, then 100 mu L of 10% methanol solution is used to balance the enrichment material, 10 mu g of phosphorylated protein alpha-casein and 10mg of interference bovine serum albumin BSA are dissolved in 10mL of 10% methanol solution for sample loading, the mixture and the enrichment material are mixed and incubated, the oscillation speed of incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining pellet was rinsed with 10mL of 15% methanol solution and shaken at 2000rpm for 5 minutes. And after shaking, centrifuging to remove supernatant, eluting the rest precipitate with 50 mu L of 10% ammonia water solution, shaking at 2500rpm for 10 min, and centrifuging to obtain supernatant to obtain phosphorylated protein.
Example 16
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. The method comprises the steps of loading 15mg of multi-hydrogen bond responsive polymer enrichment material into a centrifuge tube, flushing the enrichment material by 100 mu L of 40% acetonitrile/1% ammonia water solution, balancing the enrichment material by 200 mu L of 0.1% trifluoroacetic acid solution, dissolving 10 mu g of phosphorylated protein beta-casein and 20mg of interference bovine serum albumin BSA in 15mL of 0.1% trifluoroacetic acid solution, loading, mixing and incubating with the enrichment material, wherein the oscillation speed of incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining pellet was rinsed with 20mL of 5% acetonitrile/0.1% trifluoroacetic acid solution and shaken at 2000rpm for 5 minutes. After shaking, centrifuging to remove supernatant, eluting the rest precipitate with 50 μ L of 40% acetonitrile/1% ammonia water solution at 2500rpm for 10 min, shaking, and centrifuging to obtain supernatant to obtain phosphorylated protein.
Example 17
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. The method comprises the steps of loading 15mg of multi-hydrogen bond responsive polymer enrichment material into a centrifuge tube, flushing the enrichment material by 100 mu L of 10% ammonia water solution, balancing the enrichment material by 200 mu L of 15% acetonitrile/0.1% formic acid solution, dissolving 10 mu g of phosphorylated protein beta-casein and 20mg of interferent bovine serum albumin BSA (BSA) in 15mL of 15% acetonitrile/0.1% formic acid solution, loading the mixture, mixing the mixture with the enrichment material, incubating, wherein the incubation oscillation speed is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining precipitate was rinsed with 20mL of 15% acetonitrile/0.1% formic acid solution and shaken at 2000rpm for 5 minutes. And after shaking, centrifuging to remove supernatant, eluting the rest precipitate with 50 mu L of 10% ammonia water solution, shaking at 2500rpm for 10 min, and centrifuging to obtain supernatant to obtain phosphorylated protein. The enrichment results are shown in FIG. 5. The mass spectrum of the eluent has only the signal of phosphorylated protein beta-casein and no signal of non-phosphorylated protein BSA, which shows that when the non-phosphorylated protein of the interferent is increased to 2000 times (mass ratio) of the phosphorylated protein, the multi-hydrogen bond responsive polymer enrichment material can still be selectively enriched to the phosphorylated protein from the complex biological sample mixture.
Example 18
And taking the multi-hydrogen bond responsive polymer as an enrichment material to enrich the phosphorylated protein in the dSPE mode. The method comprises the steps of loading 15mg of multi-hydrogen bond responsive polymer enrichment material into a centrifuge tube, flushing the enrichment material by 100 mu L of 30% ethanol/1% ammonia water solution, then balancing the enrichment material by 200 mu L of 20% ethanol/acetic acid-ammonium acetate (the concentration of buffer salt is 10mmol/mL), dissolving 10 mu g of phosphorylated protein alpha-casein and 20mg of interferent bovine serum albumin BSA in 15mL of 20% ethanol/acetic acid-ammonium acetate (the concentration of buffer salt is 10mmol/mL) buffer solution, loading the mixture, mixing the mixture with the enrichment material, and incubating, wherein the oscillation rotating speed of incubation is 1000rpm, the incubation time is 30 minutes, and the incubation temperature is 20 ℃. After incubation, the supernatant was discarded by centrifugation, and the remaining pellet was rinsed with 20mL of 25% ethanol/acetic acid-ammonium acetate buffer (buffer salt concentration 10mmol/mL) and shaken at 2000rpm for 5 minutes. After shaking, centrifuging to remove supernatant, eluting the rest precipitate with 50 μ L30% ethanol/1% ammonia water solution at 2500rpm for 10 min, shaking, and centrifuging to obtain supernatant to obtain phosphorylated protein.
Claims (7)
1. A phosphorylated protein enrichment method based on a multi-hydrogen bond responsive polymer is characterized in that: taking a multi-hydrogen bond responsive polymer as a phosphorylated protein enrichment material, and adopting a column solid phase extraction mode (SPE) or a dispersed solid phase extraction mode (dSPE) to enrich and separate phosphorylated protein in a biological sample;
the multi-hydrogen bond responsive polymer is obtained by copolymerizing isopropyl acrylamide and thiourea derivatives on the surface of a substrate material by utilizing a surface initiation-atom transfer radical polymerization method, and has a molecular structure shown as follows:
wherein x is 0.1-0.9; n-102～106(ii) a R is amino acid, monosaccharide, disaccharide, dipeptide, oligopeptide or zwitterionic compound with the number of amino acids of 2-4; matrix is a base material.
2. The enrichment process of claim 1, wherein: the multi-hydrogen bond responsive polymer is used as a phosphorylated protein enrichment material, and the specific steps of enriching and separating phosphorylated protein by adopting a column solid phase extraction mode (SPE) or a dispersed solid phase extraction mode (dSPE) are as follows:
in the SPE mode, firstly, loading a multi-hydrogen bond responsive polymer material serving as a phosphorylated protein enrichment material onto a pipette tip with a sieve plate at the tail end or an SPE small column (tip), flushing the enrichment material by adopting eluent, then balancing the enrichment material by using sample liquid, then loading a biological sample dissolved in the sample liquid onto the enrichment material, then leaching the enrichment material by adopting leacheate to remove non-phosphorylated protein, and finally eluting the phosphorylated protein on the enrichment material by using the eluent to obtain the phosphorylated protein;
in the dSPE mode, a multi-hydrogen bond responsive polymer material is used as a phosphorylated protein enrichment material and placed in a centrifuge tube, an eluent is used for washing the enrichment material, then a sample liquid is used for balancing the enrichment material, then the enrichment material is mixed with the sample liquid dissolved with a biological sample for incubation, after incubation is completed, supernatant is discarded through centrifugation, a residual precipitate part is rinsed and vibrated through a rinsing liquid, after rinsing and vibrating, supernatant is discarded through centrifugation, a residual precipitate part is eluted and vibrated through the eluent, and after elution and vibrating, the phosphorylated protein is obtained through centrifugation and supernatant taking.
3. The method of claim 2, wherein:
the sample loading solution is a mixed solution of a buffer solution prepared from acid and buffer salt and an organic solvent, the volume ratio of the organic solvent is 0-50%, the mass concentration of the acid is 0-5%, the concentration of the buffer salt is 0-200mM, the pH value of the sample loading solution is within the range of 1-7, and when a biological sample is dissolved in the sample loading solution and loaded on an enrichment material, the proportional relation between the biological sample and the sample loading solution is that 0.01-1 mu g of biological sample is dissolved in each 1 mu L of the sample loading solution;
the leacheate consists of a mixed solution of a buffer solution prepared from acid and buffer salt and an organic solvent, wherein the volume ratio of the organic solvent is 0-50%, the mass concentration of the acid is 0-5%, the concentration of the buffer salt is 0-200mM, and the pH value of the leacheate is within the range of 1-7;
the eluent is composed of a mixed solution of a buffer solution and an organic solvent, the volume ratio of the organic solvent is 0-50%, the concentration of buffer salt is 0-200mM, and the pH value of the eluent is within the range of 7-12.
4. A method according to any of claims 2 or 3, characterized by: the experimental operating temperature is 15-50 ℃.
5. A method according to any of claims 2 or 3, characterized by: when the SPE mode is used to enrich phosphorylated proteins:
the volume of eluent used for washing the enrichment material is 2-50 times of the volume of the enrichment material;
the volume of the sample loading liquid used for balancing the enrichment material is 2-50 times of the volume of the enrichment material;
the volume of the sample loading liquid containing the biological sample is 2-100 times of the volume of the enrichment material;
the volume of the leacheate used for leaching the enrichment material is 2-100 times of the volume of the enrichment material;
the volume of the eluent used for eluting the phosphorylated protein is 2 to 30 times of the volume of the enrichment material;
when the dSPE mode was used to enrich for phosphorylated proteins:
the volume of eluent used for washing the enrichment material is 3-500 times of the volume of the enrichment material;
the volume of the sample loading liquid used for balancing the enrichment material is 3-500 times of the volume of the enrichment material;
the volume of the sample loading liquid containing the biological sample is 3-2000 times of the volume of the enrichment material;
the volume of the leacheate for leaching the enrichment material is 3-3000 times of the volume of the enrichment material;
the volume of the eluent used for eluting phosphorylated protein is 5 to 200 times of the volume of the enrichment material.
6. The method of claim 2, wherein: when the dSPE mode is adopted to enrich phosphorylated proteins, the oscillation revolution of incubation is 100-.
7. A method according to any of claims 2 or 3, characterized by: the organic solvent is one or more of acetonitrile, methanol and ethanol; the buffer salt is one of ammonium formate, ammonium acetate, ammonium bicarbonate, Tris (hydroxymethyl) aminomethane (Tris) and phosphate buffer salt solution; the acid is one or more of formic acid, acetic acid and trifluoroacetic acid.
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