CN114272915A - Phosphine group ionic liquid modified nano composite material, preparation method thereof and application thereof in enrichment of phosphorylated peptide - Google Patents
Phosphine group ionic liquid modified nano composite material, preparation method thereof and application thereof in enrichment of phosphorylated peptide Download PDFInfo
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Abstract
A phosphine group ionic liquid modified nano composite material and a preparation method thereof are disclosed, and the material is applied to the enrichment of phosphorylated peptides in biological samples containing phosphorylated peptides. The method comprises the following steps: (1) carrying out substitution reaction on a substrate material modified by chloropropyl and Polyethyleneimine (PEI), and fixing the PEI on the surface of the substrate material; (2) carrying out quaternization reaction on the brominated alkyl phosphonate and the PEI layer to obtain the ionic liquid modification layer with the organic phosphine function. (3) The metal ions are fixed on the surface of the material through ester group hydrolysis reaction and metal ion complexing reaction. The functional material prepared by the invention has larger specific surface area, excellent hydrophilicity, more metal solid loading capacity, excellent sensitivity, high specific selectivity, size exclusion effect and excellent reusability. The nano composite material is suitable for enrichment and purification of phosphorylated peptides of complex biological samples. The material has wide application prospect in the fields of proteomics and biomedicine.
Description
Technical Field
The invention belongs to the field of functional materials and life science, and particularly relates to a phosphine ionic liquid modified nano composite material, and a preparation method and application thereof in enrichment of phosphorylated peptide.
Background
In the 90 s of the 20 th century, with the rapid development of genomics, the human genome sequencing work was almost completed. It is initially recognized that studies based on mRNA levels do not contain all information of life, and therefore direct performer-protein studies on life activities are becoming a focus. Statistically, more than 30% of proteins are phosphorylated in mammalian cells to varying degrees. Protein phosphorylation is the most widespread post-translational modification in nature, and different post-translational modifications play different roles in the regulation of vital functions, such as playing a key regulatory role in signaling, immune response, cell differentiation and apoptosis (Pawson T, Scott J d. trends Biochem Sci, 2005, 30(6): 286). The comprehensive understanding of protein phosphorylation has important biological significance and clinical application value for disease diagnosis and pathology research. In recent years, mass spectrometry technology has been rapidly developed and has also been widely applied to the field of proteomics research. In complex biological samples, a large amount of non-phosphorylated peptides exist, but the content of phosphorylated proteins is very small and ionization efficiency in mass spectrometry is low, so that low-abundance phosphorylated peptide signals are inhibited. Therefore, before mass spectrometry, designing an enrichment material for efficiently capturing phosphorylated peptides is a key for accurately identifying information of the phosphorylated peptides.
The traditional methods for enriching phosphorylated peptides include immunoaffinity chromatography, metal oxide affinity chromatography, immobilized metal ion affinity chromatography, and the like. At present, various functional materials are synthesized to capture phosphorylated peptides or phosphorylated proteins according to different mechanisms of phosphorylated peptide enrichment, and the traditional methods solve the problem of phosphorylated peptide enrichment, but some problems still cannot be well avoided, such as weak anti-interference, low detection sensitivity, low selectivity, poor capability of eliminating biomacromolecule interference and the like.
Disclosure of Invention
Based on the problems in the prior art, the invention introduces a new precursor Polyethyleneimine (PEI) to improve the nitrogen content of the material surface, and then obtains the organic phosphorus functional ionic liquid with high specific content by quaternary amination and crosslinking of halogenated alkyl organic phosphate and PEI, which is beneficial to improving the metal ion content, thereby improving the enrichment capacity, sensitivity and selectivity of the enrichment material to phosphorylated peptides.
In order to solve the above technical problems, the present invention is solved by the following technical solutions.
The phosphine group ionic liquid modified nano composite material is prepared by the following method: (1) dispersing the substrate material A in anhydrous toluene, adding 3-chloropropyltriethoxysilane, and reacting with N2Continuously stirring and heating in the atmosphere to obtain a chloropropyl modified nano material, namely A-CP, washing and drying; the substrate material A is nano silicon dioxide (nSiO)2) Magnetic core-shell structure nano silicon dioxide (Fe)3O4@nSiO2) Or mesoporous silica-coated graphene (G @ mSiO)2) Any one of the above; (2) dissolving PEI in absolute ethyl alcohol, stirring uniformly, adding the obtained material A-CP into the solution, stirring, heating, and grafting a layer of functional material with polyethyleneimine on the surface of the A-CP, namely A @ PEI; (3) the obtained material A @ PEI is dispersed in anhydrous toluene, and then excessive bromo alkyl phosphate is added into the anhydrous toluene, and the mixture is stirred and heated for reaction. Washing and drying to obtain the phosphine group functionalized ionic liquid modified material, namely A @ PEI-PFIL; the alkyl phosphate bromide is any one of the following 3: diethyl (3-bromopropyl) phosphate, diethyl (4-bromobutyl) phosphate and diethyl (5-bromopentyl) phosphate; (4) dispersing the obtained material A @ PEI-PFIL in concentrated hydrobromic acid diluted by 2 times by deionized water, stirring and heating, neutralizing by using a NaOH solution (PH is 11), and drying; (5) dispersing the material obtained in the step (4) in a metal salt solution, reacting for 2h at 37 ℃ to obtain an affinity material for fixing metal ions, washing and drying to obtain the nano material A @ PEI-PFIL-Mn+(Mn+＝Ti4+,Ga3+) (ii) a The metal salt solution is Ti (SO)4)2Or GaCl3。
The preparation method of the phosphine group ionic liquid modified nano composite material comprises the following steps:
(1) dispersing the substrate material A in anhydrous toluene, adding 3-chloropropyltriethoxysilane, and adding N2Continuously stirring and heating under the atmosphere to obtain a chloropropyl modified nano material, namely A-CP, washing and drying; the substrate material A is nano silicon dioxide (nSiO)2) Magnetic core-shell structure nano silicon dioxide (Fe)3O4@nSiO2) Or mesoporous silica-coated graphene (G @ mSiO)2) Any of the above. (2) Dissolving PEI in absolute ethyl alcohol, stirring uniformly, adding the obtained material A-CP into the solution, stirring, heating, and grafting PEI onto the surface of the obtained A-CP to obtain a material A @ PEI; (3) the obtained material A @ PEI is dispersed in anhydrous toluene, and then excessive bromo alkyl phosphate is added into the anhydrous toluene, and the mixture is stirred and heated for reaction. Washing and drying to obtain a phosphine functionalized ionic liquid modified material, namely A @ PEI-PFIL; the alkyl phosphate bromide is any one of the following 3: diethyl (3-bromopropyl) phosphate, diethyl (4-bromobutyl) phosphate and diethyl (5-bromopentyl) phosphate; (4) dispersing the obtained material A @ PEI-PFIL in concentrated hydrobromic acid diluted by 2 times by deionized water, stirring and heating, neutralizing by using a NaOH solution (PH is 11), and drying; (5) dispersing the material obtained in the step (4) in a metal salt solution, and reacting for 2h at 37 ℃ to obtain the immobilized metal ion affinityAnd mixing the materials, washing and drying to obtain the nano material A @ PEI-PFIL-Mn+(Mn+＝Ti4+,Ga3+) (ii) a The metal salt solution is Ti (SO)4)2Or GaCl3。
Further, in the step (1), the reaction temperature is 85 ℃, and the reaction time is 14 h.
Further, in the step (2), the reaction temperature is 80 ℃, and the reaction time is 24 h.
Further, in the step (3), the reaction temperature is 110 ℃, and the reaction time is 16 h.
Further, the washing solutions in the steps (1), (2) and (3) are all ethanol.
The invention discloses application of a phosphine based ionic liquid modified nano composite material in enrichment of phosphorylated peptides, wherein the phosphine based ionic liquid modified nano composite material is used for enriching the phosphorylated peptides.
The principle of the invention is as follows: the preparation method comprises the steps of quaternizing a polyethyleneimine modified nano material by using brominated alkyl phosphate, crosslinking organic phosphorus functional ionic liquid, and modifying the organic phosphorus functional ionic liquid on a substrate material to obtain the polyethyleneimine modified nano material, namely A @ PEI-PFIL; after the acidification treatment, the organic phosphoric groups are modified with metal ions (M)n+) Obtaining A @ PEI-PFIL-Mn+Fixing the metal ion affinity chromatography material.
The invention changes the combination of the substrate material A and the brominated alkyl phosphate ester, namely: selecting nano-silica, silica-coated magnetic sphere nano-particles, mesoporous silica-coated graphene and diethyl (3-bromopropyl) phosphate nano-composite materials, preparing nine IMAC adsorbents of different substrates and different ligand materials, namely A @ PEI-PFIL-M, from the nano-silica, the silica-coated magnetic sphere nano-particles, the mesoporous silica-coated graphene and diethyl (4-bromobutyl) phosphate nano-composite materials, and the nano-silica, the silica-coated magnetic sphere nano-particles, the mesoporous silica-coated graphene and diethyl (5-bromopentyl) phosphate nano-composite materialsn+(wherein, A ═ nSiO2、Fe3O4@nSiO2Or G @ mSiO2PFIL is prepared from diethyl (3-bromopropyl) phosphate, diethyl (4-bromobutyl) phosphate or diethyl (5-bromopentyl) phosphate, Mn+＝Ti4+,Ga3+)。
Compared with the prior art, the invention has the following beneficial effects: (1) the modification process is simple, the operation is easy, the appearance of the substrate material cannot be changed, and the prepared material has good environmental stability and reusability. (2) The fixed metal ion affinity material synthesized in the invention: a @ PEI-PFIL-Mn+(Mn+＝Ti4+,Ga3+) The material is an IMAC type adsorbing material, can be used for specific enrichment of phosphorylated peptide by utilizing the affinity of phosphorylated peptide and fixed metal ions, effectively solves the problems of insufficient enrichment selectivity, low adsorption specific capacity, poor protein exclusion effect and the like of the synthesized material, and can be used for enrichment of phosphorylated peptide in a complex biological sample.
Drawings
FIG. 1 shows a nanocomposite A @ PEI-PFIL-Mn+(wherein, A ═ nSiO2、Fe3O4@nSiO2Or G @ mSiO2(ii) a PFIL is prepared from diethyl (3-bromopropyl) phosphate, diethyl (4-bromobutyl) phosphate or diethyl (5-bromopentyl) phosphate, Mn+＝Ti4+,Ga3+) Synthetic flow diagram.
FIGS. 2 a-2 j are mass spectra of beta-casein hydrolysate (200 fmol/. mu.L); phosphorylated peptide signals are denoted by #, dephosphorylated residues are denoted by #, where:
FIG. 2a is a direct detection map of a beta-casein enzymatic hydrolysate;
FIG. 2b shows the beta-casein enzymatic hydrolysate being subjected to nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (5-bromopentyl) phosphate) mass spectrum after treatment;
FIG. 2c shows the beta-casein enzymatic hydrolysate passing through nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (4-bromobutyl) phosphate) mass spectrum after treatment;
FIG. 2d shows the beta-casein enzymatic hydrolysate passing through nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (3-bromopropyl) phosphate) mass spectrum after treatment;
FIG. 2e shows the beta-casein enzymatic hydrolysate being Fe3O4@ nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (5-bromopentyl) phosphate) mass spectrum after treatment;
FIG. 2f shows the beta-casein enzymolysis solution passing through Fe3O4@ nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (4-bromobutyl) phosphate) mass spectrum after treatment;
FIG. 2g shows the beta-casein enzymolysis solution passing through Fe3O4@ nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (3-bromopropyl) phosphate) mass spectrum after treatment;
FIG. 2h shows the beta-casein enzymatic hydrolysate passing through G @ mSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (5-bromopentyl) phosphate) mass spectrum after treatment;
FIG. 2i shows the beta-casein enzymatic hydrolysate passing through G @ mSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (4-bromobutyl) phosphate) mass spectrum after treatment;
FIG. 2j shows the beta-casein enzymatic hydrolysate passing through G @ mSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (3-bromopropyl) phosphate) mass spectrum after treatment.
FIG. 3 shows G @ mSiO for beta-casein enzymatic hydrolysate2@PEI-PFIL-Ga3+A mass spectrogram after treatment; PFIL ═ diethyl (3-bromopropyl) phosphate, phosphorylated peptide signals are indicated by x, and dephosphorylated residues are indicated by #.
FIGS. 4 a-4 b are mass spectrograms of enzymatic hydrolysis mixed solution (molar ratio is 1:5000) of beta-casein and bovine serum albumin BSA; PFIL ═ diethyl (3-bromopropyl) phosphate, phosphorylated peptide signals are indicated by x, and dephosphorylated residues are indicated by #; wherein:
FIG. 4a is G @ mSiO2@PEI-PFIL-Ga3+A mass spectrogram after treatment;
FIG. 4b is G @ mSiO2@PEI-PFIL-Ti4+And (4) a mass spectrum after treatment.
FIG. 5 shows the enzymatic hydrolysis of a mixture of beta-casein (1.43pmol) and bovine serum albumin BSA (molar ratio 1: 12000) at G @ mSiO2@PEI-PFIL-Ti4+A mass spectrogram after treatment; PFIL ═ diethyl (3-bromopropyl) phosphate, phosphorylated peptide signals indicated by x,the dephosphorylated residue is indicated by #.
FIGS. 6 a-6 d are mass spectra of saliva; PFIL ═ diethyl (3-bromopropyl) phosphate, phosphorylated peptide signals are indicated by a sum and plus dot number or conventional numbers; wherein:
FIG. 6a is a mass spectrum of a saliva sample directly analyzed;
FIG. 6b is nSiO2@PEI-PFIL-Ti4+A mass spectrogram after treatment;
FIG. 6c is Fe3O4@nSiO2@PEI-PFIL-Ti4+A mass spectrogram after treatment;
FIG. 6d is G @ mSiO2@PEI-PFIL-Ti4+And (4) a mass spectrum after treatment.
FIGS. 7 a-7 b are adsorbents G @ mSiO2@PEI-PFIL-Ti4+A mass spectrum of protein exclusion; PFIL ═ diethyl (3-bromopropyl) phosphate, phosphorylated peptide signals are indicated by x, and dephosphorylated residues are indicated by #; wherein:
FIG. 7a is β -casein: a mass spectrum of BSAprotein at 1:1000: 1000;
fig. 7b is a mass spectrum of β -casein: β -caseinprotein: BSAprotein ═ 1:2000: 2000.
FIGS. 8a to 8c are mass spectra of the beta-casein hydrolysate; phosphorylated peptide signals are indicated by #, dephosphorylated residues are indicated by #; wherein:
FIG. 8a is G @ mSiO2@PEI-PFIL-Ti4+Mass spectrogram of 1 st enrichment of the beta-casein enzymolysis liquid;
FIG. 8b is G @ mSiO2@PEI-PFIL-Ti4+Repeatedly using the beta-casein enzymolysis liquid for 5 times to obtain an enriched mass spectrogram;
FIG. 8c is G @ mSiO2@PEI-PFIL-Ti4+And (3) repeatedly using the beta-casein hydrolysate for 10 times of enriched mass spectrograms.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
The embodiments described below by referring to the drawings, in which the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout, are exemplary only for explaining the present invention, and are not construed as limiting the present invention.
Example 1: wherein A ═ nSiO2PFIL ═ diethyl (5-bromopentyl) phosphate.
Example 2: wherein A ═ nSiO2PFIL ═ diethyl (4-bromobutyl) phosphate.
Example 3: wherein A ═ nSiO2PFIL ═ diethyl (3-bromopropyl) phosphate.
Example 4: wherein A ═ Fe3O4@nSiO2PFIL ═ diethyl (5-bromopentyl) phosphate.
Example 5: wherein A ═ Fe3O4@nSiO2PFIL ═ diethyl (4-bromobutyl) phosphate.
Example 6: wherein A ═ Fe3O4@nSiO2PFIL ═ diethyl (3-bromopropyl) phosphate.
Example 7: wherein A ═ G @ mSiO2PFIL ═ diethyl (5-bromopentyl) phosphate.
Example 8: wherein A ═ G @ mSiO2PFIL ═ diethyl (4-bromobutyl) phosphate.
Example 9: wherein A ═ G @ mSiO2PFIL ═ diethyl (3-bromopropyl) phosphate.
In the above 9 embodiments, the difference is that the base material a and the ligand PFIL are different, and the preparation method is the same: preparing a chloropropyl modified nano composite to obtain A-CP, grafting polyethyleneimine on the surface of the prepared nano composite, performing crosslinking quaternization on bromoalkyl phosphate and the polyethyleneimine, and modifying the surface of the nano composite to obtain A @ PEI-PFIL; after acidizing, metal ions are fixed on the organic phosphate group to obtain the fixed metal ion affinity adsorbent A @ PEI-PFIL-Mn+。
The preparation method comprises the following steps:
(1) the preparation method of the chloropropyl modified nano material comprises the following steps: dispersing 200mg of a substrate material in 30mL of anhydrous toluene, carrying out ultrasonic treatment for 10 minutes, adding 0.6mL of 3-chloropropyltriethoxysilane, heating to 85 ℃ under the protection of nitrogen, and carrying out stirring reaction for 14 hours. After the reaction is finished, centrifugally separating the solid, washing the solid with ethanol for several times, and drying the washed solid to obtain the A-CP.
(2) Preparation of A @ PEI: 1g of polyethyleneimine is dissolved in 25mL of absolute ethanol, stirred for 10 minutes, 200mg of A-CP is dispersed in the absolute ethanol solution of polyethyleneimine, ultrasonic treatment is carried out for 10 minutes, the solution is heated to 80 ℃, and stirring reaction is carried out for 24 hours. And after the reaction is finished, centrifugally separating the solid, washing the solid for several times by using deionized water and ethanol in sequence, and drying to obtain the A @ PEI.
(3) Preparation of A @ PEI-PFIL: 0.5g of brominated alkylphosphate was dropped into 15mL of anhydrous toluene, sonicated for ten minutes, 100mg of A @ PEI was dispersed in an anhydrous toluene solution containing brominated alkylphosphate, sonicated for 10 minutes, and magnetically stirred in an oil bath at 110 ℃ for 16 hours. And after the reaction is finished, centrifugally separating solids, washing the solids for several times by using ethanol, and drying the solids to obtain the A @ PEI-PFIL.
(4) Acidification treatment of A @ PEI-PFIL: the resulting A @ PEI-PFIL was dispersed in 6mL of concentrated hydrobromic acid diluted 2-fold and magnetically stirred in an oil bath at 110 ℃ for 2 h. After the reaction is finished, neutralizing the solution by using a sodium hydroxide solution (pH is 11), washing the solution to be neutral by using deionized water, and drying the solution to obtain the sodium salt A @ PEI-PFIL-Na+。
(5)A@PEI-PFIL-Mn+The preparation of (1): treating the acid-treated A @ PEI-PFIL-Na+Dispersing in 15 mL0.1M metal salt solution, and shaking at room temperature for 2 h. After the reaction is finished, centrifugally separating solids, washing the solids by deionized water and ethanol in sequence, and drying the solids at the temperature of 85 ℃ to obtain the A @ PEI-PFIL-Mn+. The metal salt solution is Ti (SO)4)2Or GaCl3And (3) solution.
The experimental tests and the description of the figures are as follows:
(1) in order to investigate the use of 3 different substrates and 3 different ligands for nanomaterials in modifying titanium ions (A @ PEI-PFIL-Ti)4+) The effect of the enrichment of phosphorylated peptides was later determined to determine the effect of different substrate materials on enrichment, and we compared nine A @ PEI-PFIL-Ti4+Adsorbent pairAnd (3) the enrichment effect of the phosphorylated peptide in the standard protein beta-casein hydrolysate.
5mg of beta-casein was dissolved in 1mL of 25mM ammonium bicarbonate buffer solution (pH 8); adding trypsin (the mass ratio of the trypsin to the substrate is 1:50) into the mixed solution, and reacting for 12 hours at 37 ℃. And storing the product after enzymolysis in a refrigerator at the temperature of minus 20 ℃ for later use.
To compare the IMAC adsorbent A @ PEI-PFIL-Ti of nine different substrates4+For the enrichment effect of phosphorylated peptides, we first selected the standard protein β -casein as the enriched sample.
2mg of the adsorbents of 3 different substrates and 3 different ligands are weighed into 0.8mL of enrichment buffer (50% ACN, 0.1% TFA, v/v), and after 10 minutes of ultrasonic treatment, 100. mu.L of the dispersion is taken out for enrichment experiment, and 1. mu.L of standard peptidase hydrolyzed solution (200 fmol/. mu.L) is added into 100. mu.L of the dispersion. Then, the mixture was placed in a constant temperature metal bath, shaken at 37 ℃ for 30min, centrifuged or magnetically separated to remove the solid and the solid material was washed three times with an enrichment buffer. Finally, the washed solid material was dispersed with 10. mu.L of 0.4M aqueous ammonia, shaken at 37 ℃ for 15min, centrifuged for three minutes, 5. mu.L of the supernatant was mixed with 5. mu.L of a matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. mu.L of the mixture was dropped onto a target plate of MALDI, dried in air, and subjected to MALDI-TOF MS analysis.
The mass spectrometry results are shown in the series of fig. 2, fig. 2a is the result of directly performing mass spectrometry without processing the sample, and it can be seen from fig. 2a that there is almost no signal of phosphorylated peptide; the analysis results obtained after the sample was treated with nine adsorbents are shown in fig. 2 b-j, the sample was subjected to nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (5-bromopentyl) phosphate), nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (4-bromobutyl) phosphate), nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (3-bromopropyl) phosphate), Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (5-bromopentyl) phosphate), Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl)(4-bromobutyl) phosphate), Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (3-bromopropyl) phosphate), G @ mSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (5-bromopentyl) phosphate), G @ mSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (4-bromobutyl) phosphate) and G @ mSiO2@PEI-PFIL-Ti4+(PFIL ═ diethyl (3-bromopropyl) phosphate) treatment detected 4, 3, 5, 2, 4, 3, 5 and 5 phosphorylated peptide signal peaks and corresponding 1, 0, 1, 2 and 2 dephosphorylated residue signal peaks, respectively. The mass spectrum result shows that the signal peak intensity of the phosphorylated peptide is large, the background is clean, and the experimental result shows that the nine adsorbents can effectively enrich the phosphorylated peptide; comprehensive analysis of the number of phosphorylated peptide signals and the appearance of hetero-peaks, as G @ mSiO2Adsorbent G @ mSiO as substrate, ligand PFIL ═ diethyl (3-bromopropyl) phosphate2@PEI-PFIL-Ti4+The reason why the mesoporous silica has better specificity is probably that more hydrophilic polyethyleneimine can be modified due to the large specific surface area of the mesoporous silica, and more phosphorus-oxygen groups are modified to fix more metal ions, so that more phosphorylated peptides are enriched.
(2) To investigate the effect of different metal ions on enrichment of phosphorylated peptides, we will use different metal ions (Ga)3+、Ti4+) Immobilization on a substrate G @ mSiO2Ligand PFIL ═ diethyl (3-bromopropyl) phosphate, comparative immobilization of different metal ion materials G @ mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+) The enrichment effect on the phosphorylated peptide in the beta-casein hydrolysate.
Respectively weighing 2mg of two adsorbents G @ mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+) After sonication in 0.8mL of enrichment buffer (50% ACN, 0.1% TFA, v/v), 100. mu.L of the dispersion was taken for enrichment experiments and 1. mu.L of standard peptidase hydrolyzed solution (200 fmol/. mu.L) was added to the dispersion. Then, the mixture was placed in a constant temperature metal bath and shaken at 37 ℃ for 30 min. After the reaction is finished, the solid is centrifugally separated and the enrichment buffer solution is usedThe solid material was washed three times. Finally, the washed solid material was dispersed with 10. mu.L of 0.4M aqueous ammonia, shaken at 37 ℃ for 15min, centrifuged, and 5. mu.L of the supernatant was taken, mixed with 5. mu.L of a matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. mu.L of the mixture was dropped onto a target plate of MALDI, dried in air, and subjected to MALDI-TOF MS analysis.
The comparison of mass spectrometry is shown in fig. 3 and fig. 2 j. The sample is respectively passed through two adsorbents G @ mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+) After treatment, it can be observed that 1 phosphorylated peptide signal peak is detected in FIG. 3, and the background baseline is relative to the material G @ mSiO2@PEI-PFIL-Ti4+(FIG. 2j) higher, sample by G @ mSiO2@PEI-PFIL-Ti4+After the treatment (FIG. 2j), 5 signal peaks of phosphorylated peptide can be detected, and the material G @ mSiO is known from the spectrum background and the number of the detected signal peaks of phosphorylated peptide2@PEI-PFIL-Ti4+The poly-phosphorylated peptide can be better enriched.
(3) To further study the effect of different metal ions on specific enrichment of phosphorylated peptides, materials G @ mSiO immobilized with different metal ions were compared2@PEI-PFIL-Mn+(Ga3+、Ti4+) The enrichment effect on the phosphorylated peptide in the enzymolysis mixed liquid (the molar ratio is 1:5000) of the beta-casein and bovine serum albumin BSA.
Dissolving 1mg bovine serum albumin in 0.1mL 50mM ammonium bicarbonate denaturation buffer (containing 8M urea), adding 0.2mL 0.1M Dithiothreitol (DTT) solution after denaturation, reacting at 37 ℃ for 30min to reduce disulfide bonds in the protein, then adding 0.2mL 0.2M Iodoacetamide (IAA) solution, and reacting at room temperature in the dark for 30min to alkylate the reduced sulfhydryl; the product was diluted to 1mL with 50mM ammonium bicarbonate buffer (pH 8.3); adding trypsin (the mass ratio of the trypsin to the substrate is 1:50) into the mixed solution, and reacting for 16h at 37 ℃. And storing the product after enzymolysis in a refrigerator at the temperature of minus 20 ℃ for later use.
Respectively weighing 2mg of two adsorbents G @ mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+) Slow enrichment in 1mLAfter sonication in a wash (50% ACN, 0.1% TFA, v/v), 100. mu.L of the dispersion was taken for enrichment experiments and 100. mu.L of the proteolytic digestion mixture was added to the dispersion. Then, the mixture was placed in a constant temperature metal bath, shaken at 37 ℃ for 30min, centrifuged to separate the solid and washed the solid material three times with enrichment buffer. After completion of the reaction, the washed solid material was dispersed in 10. mu.L of 0.4M aqueous ammonia, shaken at 37 ℃ for 15min, centrifuged, and 5. mu.L of the supernatant was taken, mixed with 5. mu.L of a matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. mu.L of the mixture was dropped onto a target plate of MALDI, dried in the air, and subjected to MALDI-TOF MS analysis.
The results of the analysis are shown in FIG. 4 series, FIG. 4a is G @ mSiO2@PEI-PFIL-Ga3+After sample treatment, 5 phosphorylated peptide signal peaks could be detected, but some non-phosphorylated peptide signal was present, and the baseline was higher; FIG. 4b is G @ mSiO2@PEI-PFIL-Ti4+After treatment of the samples, signals for 5 phosphorylated peptides and 1 dephosphorylated residue were observed, with less non-phosphorylated peptides present and a relatively clean background. Therefore, the enrichment results from two enriched samples were analyzed together, G @ mSiO2@PEI-PFIL-Ti4+Has better specificity to the enrichment of phosphorylated peptide.
(4) To better evaluate G @ mSiO2@PEI-PFIL-Ti4+The adsorption capacity of the nano-composite to phosphorylated peptide, namely, more complex proteolytic enzyme solution is selected as an adsorption sample, namely, the molar ratio (the molar ratio is 1: 12000) of BSA enzymatic solution in a beta-casein and bovine serum albumin BSA enzymatic hydrolysis mixed solution is continuously increased.
Weighing 2mg of G @ mSiO2@PEI-PFIL-Ti4+After sonication in 0.8mL of enrichment buffer (50% ACN, 1% TFA, v/v), 100. mu.L of the dispersion was taken for enrichment experiments, and 240. mu.L of the proteolytic mixture (where. beta. -casein content was 1.43pmol) was added to the dispersion. Then, the mixture was placed in a constant temperature metal bath, shaken at 37 ℃ for 30min, centrifuged to separate the solid and washed the solid material three times with enrichment buffer. Finally, the washed solid material was dispersed with 10. mu.L of 0.4M ammonia water and shaken at 37 ℃ for 15Min, after centrifugation, 5. mu.L of the supernatant was collected, mixed with 5. mu.L of a matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. mu.L of the mixture was dropped on a target plate of MALDI, dried in the air, and subjected to MALDI-TOF MS analysis.
The mass spectrometric results are shown in FIG. 5 after passing through G @ mSiO2@PEI-PFIL-Ti4+After enrichment, a slight increase in baseline was observed in the spectra, but 6 phosphorylated peptide signals were still observed, which dominate the overall mass spectra, and the relative intensity of the phosphorylated peptide was high, so the material G @ mSiO2@PEI-PFIL-Ti4+Has good specificity to the enrichment of phosphorylated peptides.
(5) Enrichment material A @ PEI-PFIL-Ti of 3 different substrates4+(A＝nSiO2、Fe3O4@nSiO2And G @ mSiO2) Enrichment of endogenous phosphorylated peptides in saliva; PFIL ═ diethyl (3-bromopropyl) phosphate.
Respectively weighing 2mg of three adsorbents nSiO2@PEI-PFIL-Ti4+、Fe3O4@nSiO2@PEI-PFIL-Ti4+And G @ mSiO2@PEI-PFIL-Ti4+After sonication in 0.8mL of enrichment buffer (50% ACN, 0.1% TFA, v/v), 100. mu.L of the dispersion was removed for enrichment experiments and 20. mu.L of saliva sample was added to the dispersion. Then, the mixture was placed in a constant temperature metal bath and shaken at 37 ℃ for 30 min. After the reaction was complete, the solid was centrifuged and the solid material was washed three times with enrichment buffer. Finally, the washed solid material was dispersed with 10. mu.L of 0.4M aqueous ammonia, shaken at 37 ℃ for 15min, centrifuged, and 5. mu.L of the supernatant was taken, mixed with 5. mu.L of a matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. mu.L of the mixture was dropped onto a target plate of MALDI, dried in air, and subjected to MALDI-TOF MS analysis.
The analysis result is shown in the figure 6 series, figure 6a is a mass spectrogram of a saliva sample directly subjected to mass spectrometry, and it can be observed from the figure that non-phosphorylated peptide and impurity signal peaks dominate the whole spectrogram; nSiO2@PEI-PFIL-Ti4+(FIG. 6b), Fe3O4@nSiO2@PEI-PFIL-Ti4+(FIG. 6c) and G @ mSiO2@PEI-PFIL-Ti4+(FIG. 6d) after the samples were treated with 3 adsorbents, the number of signal peaks of phosphorylated peptides detected was 25, 16 and 27, respectively, indicating that the materials of the three different substrates can be used for the enrichment of endogenous phosphorylated peptides in saliva. From the mass spectrometry results, it can be seen that fig. 6b is enriched with relatively less endogenous phosphorylated peptides and stronger signals of non-phosphorylated peptides, fig. 6c and 6d are enriched with a greater number of phosphorylated peptides, but the phosphorylated peptides enriched in fig. 6d are more clearly displayed in the mass spectrogram, and the analysis results show that the adsorbents of 3 different substrates all show better affinity for the enrichment of phosphorylated peptides, and G @ mSiO2@PEI-PFIL-Ti4+The specificity for the enrichment of endogenous phosphorylated peptides in saliva is optimal.
(6) To demonstrate the size exclusion effect of mesoporous silica based on material design, we utilized the material G @ mSiO2@PEI-PFIL-Ti4+Enrichment experiments are carried out on beta-casein enzymolysis liquid (beta-casein), beta-casein (beta-casein protein) and bovine serum albumin (BSAprotein) according to a certain mass ratio.
Weighing 2mg of adsorbent G @ mSiO2@PEI-PFIL-Ti4+After ultrasonic dispersion in 0.8mL of enrichment buffer (50% ACN, 0.1% TFA, v/v), 100. mu.L of dispersion was taken out for enrichment experiment, and 10.1. mu.L and 19.2. mu.L of mixed solutions of beta-casein enzymatic hydrolysate (beta-casein), beta-casein (beta-casein), and bovine serum albumin (BSAprotein) were added to the dispersion, respectively. Then, the mixture was placed in a constant temperature metal bath and shaken at 37 ℃ for 30 min. After the reaction was complete, the solid was centrifuged and the solid material was washed three times with enrichment buffer. Finally, the washed solid material was dispersed with 10. mu.L of 0.4M aqueous ammonia, shaken at 37 ℃ for 15min, centrifuged, and 5. mu.L of the supernatant was taken, mixed with 5. mu.L of a matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. mu.L of the mixture was dropped on a target plate of MALDI, dried in air and subjected to MALDI-TOF MS analysis;
the detection results are shown in FIG. 7 series, wherein, FIGS. 7a and 7b are the material G @ mSiO2@PEI-PFIL-Ti4+Respectively carrying out the following steps on beta-casein enzymolysis liquid (beta-casein): beta-casein (beta-caseinprotein): bovine serum albumin (BSAprotein) β -casein zymolysis ═ 1:1000:1000, beta-casein enzymatic hydrolysate (beta-casein): beta-casein (beta-caseinprotein): bovine serum albumin (BSAprotein) β -casein zymolysis ═ 1:2000:2000, it can be seen from the figure that in fig. 7a, the mass ratio is 1:1000: at 1000 f, 4 peaks of phosphorylated peptide and 2 peaks of dephosphorylated residue signal were detected, and the peak signals had large intensities. In fig. 7b, the mass ratio is 1:2000:2000, the number of phosphorylated peptide signal peaks is still not reduced, the spectrogram background is cleaner, the phosphorylated peptide peaks are dominant, and the adsorbent G @ mSiO can be obtained by result analysis2@PEI-PFIL-Ti4+Can block a large amount of proteins with large molecular weight, has good protein size exclusion effect, and has great potential in the research of endogenous phosphorylated peptides.
(7) To demonstrate the stability characteristics and reusability based on material design, we utilized the material G @ mSiO2@PEI-PFIL-Ti4+The beta-casein hydrolysate was repeatedly enriched for 10 times.
Weighing 2mg of adsorbent G @ mSiO2@PEI-PFIL-Ti4+After sonication in 800. mu.L of enrichment buffer (50% ACN, 0.1% TFA, v/v), 100. mu.L of the dispersion was taken for enrichment experiments and beta-casein enzymatic hydrolysate (200fmol) was added to the dispersion. Then, the mixture was placed in a constant temperature metal bath, shaken at 37 ℃ for 30min, centrifuged to separate the solid and washed the solid material three times with enrichment buffer. Finally, the washed solid material was dispersed with 10. mu.L of 0.4M aqueous ammonia, shaken at 37 ℃ for 15min, centrifuged, and 5. mu.L of the supernatant was taken, mixed with 5. mu.L of a matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. mu.L of the mixture was dropped on a target plate of MALDI, dried in air and subjected to MALDI-TOF MS analysis; the desorbed material was washed three times with enrichment buffer and then subjected to the next enrichment-desorption cycle under the same conditions.
The detection results are shown in FIG. 8 series, wherein, FIGS. 8a and 8b are the material G @ mSiO2@PEI-PFIL-Ti4+The detection results of the enrichment of the beta-casein enzymolysis liquid after the 1 st time and the 5 th time of repeated use are respectively shown in the figure, and the enrichment effects of the 1 st time and the 5 th time are similar; after 10 enrichment cycles (fig. 8c), 4 peak signals of phosphorylated peptides were observed in the mass spectrum compared to the results of the 1 st and 5 th assays, and no significant difference in the amount of phosphorylated peptides was detected. The results show that the material still maintains good adsorption capacity after multiple enrichment, and shows good stability and reusability.
The scope of the present invention includes, but is not limited to, the above embodiments, and the present invention is defined by the appended claims, and any alterations, modifications, and improvements that may occur to those skilled in the art are all within the scope of the present invention.
Claims (7)
1. The phosphine group ionic liquid modified nano composite material is characterized by being prepared by the following method:
(1) dispersing the substrate material A in anhydrous toluene, adding 3-chloropropyltriethoxysilane, and adding N2Continuously stirring and heating under the atmosphere to obtain a chloropropyl modified nano material, namely A-CP, and then washing and drying; the substrate material A is nano silicon dioxide (nSiO)2) Magnetic core-shell structure nano silicon dioxide (Fe)3O4@nSiO2) Or mesoporous silica-coated graphene (G @ mSiO)2) Any one of the above;
(2) dissolving PEI in absolute ethyl alcohol, stirring uniformly, adding the obtained material A-CP into the solution, stirring, heating, and grafting PEI onto the surface of the obtained A-CP to obtain a material A @ PEI;
(3) dispersing the obtained material A @ PEI in anhydrous toluene, adding excessive bromo-alkyl phosphate into the anhydrous toluene, stirring, heating for reaction, washing and drying to obtain a phosphine group functionalized ionic liquid modified material A @ PEI-PFIL; the alkyl phosphate bromide is any one of the following three types: diethyl (3-bromopropyl) phosphate, diethyl (4-bromobutyl) phosphate, diethyl (5-bromopentyl) phosphate;
(4) dispersing the obtained material A @ PEI-PFIL in concentrated hydrobromic acid diluted twice by deionized water, stirring and heating, neutralizing by using NaOH solution, and drying;
(5) dispersing the material obtained in the step (4) in a metal salt solution, reacting for 2h at 37 ℃ to obtain an affinity material for fixing metal ions, washing and drying to obtain the nano material A @ PEI-PFIL-Mn+Wherein M isn+＝Ti4+,Ga3+(ii) a The metal salt solution is Ti (SO)4)2Or GaCl3。
2. The preparation method of the phosphine based ionic liquid modified nano composite material is characterized by comprising the following steps:
(1) dispersing the substrate material A in anhydrous toluene, adding 3-chloropropyltriethoxysilane, and adding N2Continuously stirring and heating under the atmosphere to obtain a chloropropyl modified nano material, namely A-CP, washing and drying; the substrate material A is nano silicon dioxide (nSiO)2) Magnetic core-shell structure nano silicon dioxide (Fe)3O4@nSiO2) Or mesoporous silica-coated graphene (G @ mSiO)2) Any one of the above;
(2) dissolving PEI in absolute ethyl alcohol, stirring uniformly, adding the obtained material A-CP into the solution, stirring, heating, and grafting PEI onto the surface of the obtained A-CP to obtain a material A @ PEI;
(3) dispersing the obtained material A @ PEI in anhydrous toluene, then adding excessive bromo-alkyl phosphate into the anhydrous toluene, stirring, heating for reaction, washing and drying to obtain a phosphine group functionalized ionic liquid modified material, namely A @ PEI-PFIL; the alkyl phosphate bromide is any one of the following three types: diethyl (3-bromopropyl) phosphate, diethyl (4-bromobutyl) phosphate and diethyl (5-bromopentyl) phosphate;
(4) dispersing the obtained material A @ PEI-PFIL in concentrated hydrobromic acid diluted twice by deionized water, stirring and heating, neutralizing by using NaOH solution, and drying;
(5) dispersing the material obtained in the step (4) in a metal salt solution, reacting for 2h at 37 ℃ to obtain an affinity material for fixing metal ions, washing and drying to obtain the nano material A @ PEI-PFIL-Mn+(Mn+＝Ti4+,Ga3+) (ii) a The metal salt solution is Ti (SO)4)2Or GaCl3。
3. The method as claimed in claim 2, wherein the reaction temperature in step (1) is 85 ℃ and the reaction time is 14 h.
4. The method as claimed in claim 2, wherein the reaction temperature in step (2) is 80 ℃ and the reaction time is 24 h.
5. The method as claimed in claim 2, wherein the reaction temperature in the step (3) is 110 ℃ and the reaction time is 16 h.
6. The method as claimed in claim 2, wherein the washing agent used in the steps (1), (2) and (3) is ethanol.
7. The application of the phosphino ionic liquid modified nanocomposite material in the enrichment of phosphorylated peptides is characterized in that the phosphino ionic liquid modified nanocomposite material in claim 1 is used for enriching phosphorylated peptides.
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| DE102006054737A1 (en) * | 2006-11-21 | 2008-05-29 | Volkswagen Ag | Use of an ionic liquid based on fluorinated or partially fluorinated alkyl phosphorus/phosphine, phosphonic- or alkyl sulfonic-acid, or di- or triacid derivatives as an electrolyte to prepare fuel cell membrane or its electrode assemblies |
| CN111617746A (en) * | 2020-06-22 | 2020-09-04 | 宁波大学 | Polyion liquid modified nano material, preparation method thereof and application thereof in enrichment of phosphorylated peptide |
| CN111644163A (en) * | 2020-06-22 | 2020-09-11 | 宁波大学 | Tripodia ionic liquid material for enriching phosphorylated polypeptide and preparation method and application thereof |
| CN111690006A (en) * | 2020-06-22 | 2020-09-22 | 宁波大学 | Imidazolyl-based ionic liquid material, preparation method thereof and application of imidazolyl-based ionic liquid material in enrichment of phosphorylated peptide |
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| DE102006054737A1 (en) * | 2006-11-21 | 2008-05-29 | Volkswagen Ag | Use of an ionic liquid based on fluorinated or partially fluorinated alkyl phosphorus/phosphine, phosphonic- or alkyl sulfonic-acid, or di- or triacid derivatives as an electrolyte to prepare fuel cell membrane or its electrode assemblies |
| CN111617746A (en) * | 2020-06-22 | 2020-09-04 | 宁波大学 | Polyion liquid modified nano material, preparation method thereof and application thereof in enrichment of phosphorylated peptide |
| CN111644163A (en) * | 2020-06-22 | 2020-09-11 | 宁波大学 | Tripodia ionic liquid material for enriching phosphorylated polypeptide and preparation method and application thereof |
| CN111690006A (en) * | 2020-06-22 | 2020-09-22 | 宁波大学 | Imidazolyl-based ionic liquid material, preparation method thereof and application of imidazolyl-based ionic liquid material in enrichment of phosphorylated peptide |
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