CN114272915B - Phosphine-based ionic liquid modified nanocomposite, preparation method thereof and application thereof in enrichment of phosphorylated peptides - Google Patents

Abstract

A phosphine-based ionic liquid modified nanocomposite material and a preparation method thereof. The material is used for enriching phosphorylated peptides in biological samples containing phosphorylated peptides. The method comprises the following steps: (1) fixing the PEI on the surface of the base material through a substitution reaction between the base material containing chloropropyl modification and polyethyleneimine (PEI); (2) combining the brominated alkyl phosphonate with the PEI The layer undergoes a quaternization reaction to obtain an organic phosphine functionalized ionic liquid modified layer. (3) Metal ions are fixed on the surface of the material through ester group hydrolysis reaction and metal ion complexation reaction. The functionalized material prepared by the present invention has a large specific surface area, superior hydrophilicity, more metal immobilization capacity, exhibits excellent sensitivity, high specific selectivity and size exclusion effect, and excellent reusability . This type of nanocomposite material is suitable for the enrichment and purification of phosphorylated peptides in complex biological samples. The material has broad application prospects in the fields of proteomics and biomedicine.

Description

Phosphine-based ionic liquid modified nanocomposite material and its preparation method and its application in enriching phosphorylated peptides

technical field

The invention belongs to the field of functional materials and life sciences, and specifically relates to a phosphine-based ionic liquid modified nanocomposite material. At the same time, the invention also relates to a preparation method of the material and its application in enriching phosphorylated peptides.

Background technique

In the 1990s, with the rapid development of genomics, the sequencing of the human genome was basically completed. People began to realize that the research based on the mRNA level cannot contain all the information of life, so the research on protein, the direct executor of life activities, has gradually become a hot spot. According to statistics, in mammalian cells, more than 30% of proteins will be phosphorylated to varying degrees. Protein phosphorylation is the most extensive post-translational modification in nature, and different post-translational modifications play different roles in the regulation of life activities, such as key regulatory roles in signal transduction, immune response, cell differentiation and apoptosis ( Pawson T, Scott J D. Trends Biochem Sci, 2005, 30(6):286). A comprehensive understanding of protein phosphorylation has very important biological significance and clinical application value for disease diagnosis and pathology research. In recent years, mass spectrometry has developed rapidly and has been widely used in the field of proteomics research. In complex biological samples, there are a large number of non-phosphorylated peptides, but the content of phosphorylated proteins is very small and the ionization efficiency in mass spectrometry is low, which makes the signal of low-abundance phosphorylated peptides suppressed. Therefore, before mass spectrometry analysis, designing an enrichment material that efficiently captures phosphorylated peptides is the key to accurately identify phosphorylated peptide information.

Traditional methods for enriching phosphorylated peptides include immunoaffinity chromatography, metal oxide affinity chromatography, and immobilized metal ion affinity chromatography. At present, according to the different mechanisms of phosphorylated peptide enrichment, a variety of functional materials have been synthesized to capture phosphorylated peptides or phosphorylated proteins. These traditional methods have solved the problem of phosphorylated peptide enrichment, but some problems still cannot It is very good to avoid, such as weak anti-interference, low detection sensitivity, low selectivity, poor ability to exclude interference from biological macromolecules, etc.

Contents of the invention

Based on the problems existing in the prior art, the present invention introduces a new precursor polyethyleneimine (PEI) to increase the nitrogen content on the surface of the material, and then cross-links the halogenated alkyl organic phosphate and PEI through quaternization to obtain a high ratio The content of organic phosphorus functional ionic liquid is beneficial to increase the content of metal ions, thereby improving the enrichment capacity, sensitivity and selectivity of the enrichment material for phosphorylated peptides.

In order to solve the above technical problems, the present invention is solved through the following technical solutions.

The phosphine-based ionic liquid modified nanocomposite in the present application is prepared by the following method: (1) base material A is dispersed in anhydrous toluene, and 3-chloropropyltriethoxysilane is added, passed through under _N2 atmosphere Stirring and heating continuously to obtain chloropropyl-modified nanomaterials, i.e. A-CP, washing and drying; the base material A is nano silicon dioxide (nSiO ₂ ), magnetic core-shell structure nano silicon dioxide (Fe ₃ O ₄ @nSiO ₂ ) or mesoporous silica-coated graphene (G@mSiO ₂ ); (2) Dissolve PEI in absolute ethanol, stir well, and add The material A-CP obtained above, after stirring and heating, will obtain a functional material with a layer of polyethyleneimine grafted on the surface of A-CP, that is, A@PEI; (3) disperse the obtained material A@PEI in In anhydrous toluene, then add excess bromoalkyl phosphate, stir and heat the reaction. After washing and drying, the phosphino-functionalized ionic liquid modification material can be obtained, that is, A@PEI-PFIL; the bromoalkyl phosphate is any one of the following three types: diethyl (3-bromopropyl ) phosphate, diethyl (4-bromobutyl) phosphate and diethyl (5-bromopentyl) phosphate; (4) disperse the obtained material A@PEI-PFIL in deionized water and dilute 2 times In concentrated hydrobromic acid, after stirring and heating, neutralize with NaOH solution (PH=11), and dry; (5) disperse the material obtained in step (4) in metal salt solution, and react at 37°C After 2 hours, the affinity material for immobilizing metal ions can be obtained. After washing and drying, the nanomaterial A@PEI-PFIL-M ⁿ⁺ (M ⁿ⁺ =Ti ⁴⁺ , Ga ³⁺ ) can be obtained; the metal salt solution It is Ti(SO ₄ ) ₂ or GaCl ₃ .

The preparation method of phosphine-based ionic liquid modified nano composite material comprises the following steps:

(1) Disperse base material A in anhydrous toluene, add 3-chloropropyltriethoxysilane, continuously stir and heat under _N2 atmosphere to obtain chloropropyl-modified nanomaterials, namely A-CP, Washing and drying; the base material A is nano-silica (nSiO ₂ ), magnetic core-shell nano-silica (Fe ₃ O ₄ @nSiO ₂ ) or graphene coated with mesoporous silica ( Any of G@mSiO ₂ ). (2) Dissolve PEI in absolute ethanol, stir evenly, add the material A-CP obtained above to the solution, stir and heat, graft PEI to the surface of A-CP obtained above to obtain material A @PEI; (3) Disperse the obtained material A@PEI in anhydrous toluene, then add excess bromoalkyl phosphate to it, stir and heat to react. After washing and drying, the phosphino-functionalized ionic liquid modification material can be obtained, that is, A@PEI-PFIL; the bromoalkyl phosphate is any one of the following three types: diethyl (3-bromopropyl ) phosphate, diethyl (4-bromobutyl) phosphate and diethyl (5-bromopentyl) phosphate; (4) disperse the obtained material A@PEI-PFIL in deionized water and dilute 2 times In concentrated hydrobromic acid, after stirring and heating, neutralize with NaOH solution (PH=11), and dry; (5) disperse the material obtained in step (4) in metal salt solution, and react at 37°C After 2 hours, the affinity material for immobilizing metal ions can be obtained. After washing and drying, the nanomaterial A@PEI-PFIL-M ⁿ⁺ (M ⁿ⁺ =Ti ⁴⁺ , Ga ³⁺ ) can be obtained; the metal salt solution It is Ti(SO ₄ ) ₂ or GaCl ₃ .

Further, in the step (1), the reaction temperature is 85° C., and the reaction time is 14 hours.

Further, in the step (2), the reaction temperature is 80° C., and the reaction time is 24 hours.

Further, in the step (3), the reaction temperature is 110° C., and the reaction time is 16 hours.

Further, the washing solutions in steps (1), (2) and (3) are all ethanol.

In the present invention, the application of the phosphine-based ionic liquid modified nanocomposite material in enriching phosphorylated peptides: the above-mentioned phosphine-based ionic liquid modified nanocomposite material is used to enrich phosphorylated peptides.

The principle of the present invention is as follows: the present invention uses bromoalkyl phosphate to quaternize nano-materials modified by polyethyleneimine, cross-links organophosphorus functional ionic liquids, and modifies them on the base material to obtain polyethyleneimine-modified nanomaterials. A@PEI-PFIL is a permanent nanomaterial, namely A@PEI-PFIL; after acidification treatment, metal ions (M ⁿ⁺ ) are modified on the organophosphate group to obtain A@PEI-PFIL-M ⁿ⁺ immobilized metal ion affinity chromatography material.

The present invention is by changing the combination of base material A and brominated alkyl phosphate, that is: 1. select nano-silica, silica-coated magnetic sphere nanoparticles and mesoporous silica-coated graphene with diethyl ( 3-bromopropyl) phosphate nanocomposites, ② nano-silica, silica-coated magnetic sphere nanoparticles and mesoporous silica-coated graphene with diethyl (4-bromobutyl) phosphate Nanocomposite materials, ③nano-silica, silica-coated magnetic sphere nanoparticles and mesoporous silica-coated graphene and diethyl (5-bromopentyl) phosphate nanocomposites, prepared nine kinds IMAC adsorbents with different substrates and different ligand materials, that is, A@PEI-PFIL-M ⁿ⁺ (where A=nSiO ₂ , Fe ₃ O ₄ @nSiO ₂ or G@mSiO ₂ PFIL=diethyl(3- bromopropyl)phosphate, diethyl(4-bromobutyl)phosphate or diethyl(5-bromopentyl)phosphate, M ⁿ⁺ =Ti ⁴⁺ , Ga ³⁺ ).

Compared with the prior art, the present invention has the following beneficial effects: (1) The modification process of the present invention is relatively simple, easy to operate, and does not change the morphology of the substrate material, and the prepared material has good environmental stability and repeatability Usability. (2) The immobilized metal ion affinity material synthesized in the present invention: A@PEI-PFIL-M ⁿ⁺ (M ⁿ⁺ =Ti ⁴⁺ , Ga ³⁺ ) is an IMAC-type adsorption material, which utilizes phosphorylated peptides and immobilized metal ions It can be used for the specific enrichment of phosphorylated peptides. The synthesized material effectively solves the problems of insufficient enrichment selectivity, low adsorption specific capacity, and poor protein exclusion effect. It can be used for phosphorylated peptides in complex biological samples. enrichment.

Description of drawings

Fig. 1 is the nanocomposite material A@PEI-PFIL-M ⁿ⁺ (wherein, A=nSiO ₂ , Fe ₃ O ₄ @nSiO ₂ or G@mSiO ₂ ; PFIL=diethyl (3-bromopropyl) phosphate , diethyl (4-bromobutyl) phosphate or diethyl (5-bromopentyl) phosphate, M ⁿ⁺ =Ti ⁴⁺ , Ga ³⁺ ) synthesis flow chart.

Figure 2a-Figure 2j is the mass spectrum of β-casein hydrolyzate (200fmol/μL); the phosphorylated peptide signal is indicated by *, and the dephosphorylated residue is indicated by #, where:

Figure 2a is a direct detection figure of β-casein hydrolyzate;

Figure 2b is the mass spectrum of the β-casein hydrolyzate treated with nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(5-bromopentyl)phosphate);

Figure 2c is the mass spectrum of β-casein hydrolyzate treated with nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(4-bromobutyl)phosphate);

Figure 2d is the mass spectrum of the β-casein hydrolyzate treated with nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(3-bromopropyl)phosphate);

Figure 2e is the mass spectrogram of β-casein hydrolyzate treated with Fe3O4@nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(5-bromopentyl)phosphate);

Figure 2f is the mass spectrum of the β-casein hydrolyzate treated with Fe3O4@nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(4-bromobutyl)phosphate);

Figure 2g is the mass spectrum of β-casein hydrolyzate treated with Fe3O4@nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(3-bromopropyl)phosphate);

Figure 2h is the mass spectrum of the β-casein hydrolyzate treated with G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(5-bromopentyl)phosphate);

Figure 2i is the mass spectrum of β-casein hydrolyzate treated with G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(4-bromobutyl)phosphate);

Fig. 2j is the mass spectrum of β-casein hydrolyzate treated with G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(3-bromopropyl)phosphate).

Figure 3 is the mass spectrum of β-casein hydrolyzate treated with G@mSiO ₂ @PEI-PFIL-Ga ³⁺ ; PFIL=diethyl(3-bromopropyl)phosphate, phosphorylated peptide signals are indicated by * , dephosphorylated residues are represented by #.

Figure 4a-Figure 4b is the mass spectrogram of the enzymatic hydrolysis mixture of β-casein and bovine serum albumin BSA (molar ratio is 1:5000); PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signal Denote by *, dephosphorylated residues are denoted by #; where:

Figure 4a is the mass spectrum after G@mSiO ₂ @PEI-PFIL-Ga ³⁺ treatment;

Figure 4b is the mass spectrum after G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ treatment.

Figure 5 is the mass spectrogram of the enzymolysis mixture of β-casein (1.43pmol) and bovine serum albumin BSA (molar ratio is 1:12000) after G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ treatment; PFIL=two Ethyl(3-bromopropyl) phosphate, phosphorylated peptide signals are indicated by *, and dephosphorylated residues are indicated by #.

Figures 6a-6d are mass spectrograms of saliva; PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signals are represented by * and dotted numbers or regular numbers; where:

Fig. 6a is the mass spectrogram of direct analysis of saliva sample;

Figure 6b is the mass spectrum after nSiO ₂ @PEI-PFIL-Ti ⁴⁺ treatment;

Figure 6c is the mass spectrum after Fe ₃ O ₄ @nSiO ₂ @PEI-PFIL-Ti ⁴⁺ treatment;

Figure 6d is the mass spectrum after G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ treatment.

Figure 7a-Figure 7b is the mass spectrum of adsorbent G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ protein exclusion; PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signals are represented by *, Dephosphorylated residues are represented by #; where:

Figure 7a is the mass spectrum of β-casein:β-caseinprotein:BSAprotein=1:1000:1000;

Figure 7b is the mass spectrum of β-casein:β-caseinprotein:BSAprotein=1:2000:2000.

Figure 8a-Figure 8c are the mass spectrograms of β-casein hydrolyzate; phosphorylated peptide signals are indicated by *, and dephosphorylated residues are indicated by #; where:

Figure 8a is the mass spectrum of the first enrichment of β-casein hydrolyzate by G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ ;

Figure 8b is the mass spectrogram of G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ enriched β-casein hydrolyzate for 5 times;

Fig. 8c is the mass spectrum of G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ enriched β-casein hydrolyzate for 10 times.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

In the following embodiments, the same or similar symbols represent the same or similar elements or elements with the same or similar functions throughout, and the embodiments described below by referring to the accompanying drawings are exemplary, and are only used to explain the present invention, and cannot be understood To limit the present invention.

Example 1: where A=nSiO ₂ , PFIL=diethyl(5-bromopentyl)phosphate.

Example 2: where A=nSiO ₂ , PFIL=diethyl(4-bromobutyl)phosphate.

Example 3: where A=nSiO ₂ , PFIL=diethyl(3-bromopropyl)phosphate.

Example 4: where A=Fe ₃ O ₄ @nSiO ₂ , PFIL=diethyl(5-bromopentyl)phosphate.

Example 5: where A=Fe ₃ O ₄ @nSiO ₂ , PFIL=diethyl(4-bromobutyl)phosphate.

Example 6: where A=Fe ₃ O ₄ @nSiO ₂ , PFIL=diethyl(3-bromopropyl)phosphate.

Example 7: where A=G@mSiO ₂ , PFIL=diethyl(5-bromopentyl)phosphate.

Example 8: where A=G@mSiO ₂ , PFIL=diethyl(4-bromobutyl)phosphate.

Example 9: where A=G@mSiO ₂ , PFIL=diethyl(3-bromopropyl)phosphate.

Among the above 9 implementations, the difference lies in the difference between the base material A and the ligand PFIL, and the preparation method is the same: first prepare the nanocomposite modified by chloropropyl group to obtain A-CP, and connect the nanocomposite on the surface of the above prepared nanocomposite Polyethyleneimine, and then use brominated alkyl phosphate and polyethyleneimine for cross-linking and quaternization, and modify the surface to obtain A@PEI-PFIL; after acidification treatment, the metal ions are fixed on the organic phosphoric acid On the group, the immobilized metal ion affinity adsorbent A@PEI-PFIL-M ⁿ⁺ was obtained.

Concrete preparation method is as follows:

(1) Preparation of chloropropyl-modified nanomaterials, the preparation method is as follows: disperse 200mg of base material in 30mL of anhydrous toluene, after ultrasonication for 10 minutes, add 0.6mL of 3-chloropropyltriethoxy Silane, under the protection of nitrogen atmosphere, heated at 85°C, and stirred for 14h. After the reaction, the solid was separated by centrifugation, washed several times with ethanol, and dried to obtain A-CP.

(2) Preparation of A@PEI: Dissolve 1 g of polyethyleneimine in 25 mL of absolute ethanol, stir for 10 minutes, disperse 200 mg of A-CP in polyethyleneimine in absolute ethanol, and perform ultrasonication 10 minutes, heated to 80 ° C, stirring the reaction for 24h. After the reaction, the solid was separated by centrifugation, washed several times with deionized water and ethanol in turn, and dried to obtain A@PEI.

(3) Preparation of A@PEI-PFIL: 0.5 g of bromoalkyl phosphate was dropped into 15 mL of anhydrous toluene, ultrasonicated for ten minutes, and 100 mg of A@PEI was dispersed in bromoalkyl phosphate containing In anhydrous toluene solution, ultrasonication was carried out for 10 minutes, and magnetic stirring was carried out in an oil bath at 110° C. for 16 hours. After the reaction, the solid was separated by centrifugation, washed several times with ethanol, and dried to obtain A@PEI-PFIL.

(4) Acidification treatment of A@PEI-PFIL: The obtained A@PEI-PFIL was dispersed in 6 mL of 2-fold diluted concentrated hydrobromic acid, and magnetically stirred in an oil bath at 110 °C for 2 h. After the reaction, neutralize with sodium hydroxide solution (pH=11), wash with deionized water until neutral, and dry to obtain the sodium salt A@PEI-PFIL-Na ⁺ .

(5) Preparation of A@PEI-PFIL-M ⁿ⁺ : The acid-treated A@PEI-PFIL-Na ⁺ was dispersed in 15 mL of 0.1 M metal salt solution and shaken at room temperature for 2 h. After the reaction, the solid was separated by centrifugation, washed with deionized water and ethanol in sequence, and dried at 85°C to obtain A@PEI-PFIL-M ⁿ⁺ . The metal salt solution is Ti(SO ₄ ) ₂ or GaCl ₃ solution.

Experimental tests and descriptions of the accompanying drawings are as follows:

(1) In order to investigate the effect of three different substrates and three different ligands on the enrichment of phosphorylated peptides after modification of titanium ions (A@PEI-PFIL-Ti ⁴⁺ ), so as to determine the effect of different substrate materials on the enrichment of phosphorylated peptides. We compared the enrichment effect of nine kinds of A@PEI-PFIL-Ti ⁴⁺ adsorbents on the enrichment of phosphorylated peptides in the standard protein β-casein hydrolyzate.

Dissolve 5mg of β-casein in 1mL of 25mM ammonium bicarbonate buffer solution (pH=8); add trypsin to the mixed solution (the mass ratio of trypsin to substrate is 1:50), and react at 37°C for 12h . The product after enzymatic hydrolysis was stored in a refrigerator at -20°C until use.

In order to compare the enrichment effect of nine different substrate IMAC adsorbents A@PEI-PFIL-Ti ⁴⁺ on phosphorylated peptides, we first selected the standard protein β-casein as the enriched sample.

Weigh 2 mg of adsorbents with 3 different substrates and 3 different ligands into 0.8 mL of enrichment buffer (50% ACN, 0.1% TFA, v/v), and after ultrasonication for 10 minutes, take out 100 μL of the dispersion For enrichment experiments, 1 μL of standard peptidase hydrolyzate (200 fmol/μL) was added to 100 μL of the dispersion. Then, the mixture was placed in a constant temperature metal bath, shaken at 37° C. for 30 min, centrifuged or magnetically separated, and the solid material was washed three times with enrichment buffer. Finally, disperse the washed solid material with 10 μL of 0.4M ammonia water, shake at 37°C for 15 minutes, centrifuge for three minutes, take 5 μL of supernatant and 5 μL of matrix solution (saturated DHB solution, containing 50% ACN and 0.1% After TFA) mixing, 1 μL of the mixed solution was dropped on a MALDI target plate, dried in air, and analyzed by MALDI-TOF MS.

The results of mass spectrometry are shown in Figure 2 series. Figure 2a is the result of direct mass spectrometry analysis of the sample without treatment. It can be seen from Figure 2a that there is almost no signal of phosphorylated peptide; after the sample is treated with nine kinds of adsorbents, the obtained The analysis results are shown in bj in Fig. 2. The sample was passed through nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(5-bromopentyl) phosphate), nSiO ₂ @PEI-PFIL-Ti ^{4 +} (PFIL=diethyl(4-bromobutyl)phosphate), nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(3-bromopropyl)phosphate), Fe ₃ O ₄ @ nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(5-bromopentyl)phosphate), Fe ₃ O ₄ @nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(4 -bromobutyl)phosphate), Fe ₃ O ₄ @nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(3-bromopropyl)phosphate), G@mSiO ₂ @PEI-PFIL- Ti ⁴⁺ (PFIL=diethyl(5-bromopentyl)phosphate), G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL=diethyl(4-bromobutyl)phosphate) and G @mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (PFIL = diethyl(3-bromopropyl) phosphate) treatment can detect 4, 3, 5, 2, 4, 3, 5, 5 and 5 Phosphorylated peptide signal peaks and corresponding 1, 0, 1, 0, 1, 1, 1, 2, and 2 dephosphorylated residue signal peaks. From the results of mass spectrometry, it can be seen that the signal peak intensity of phosphorylated peptides is relatively strong, and the background is relatively clean. The experimental results show that these nine adsorbents can effectively enrich phosphorylated peptides; from the number of phosphorylated peptide signals and the situation of miscellaneous peaks Comprehensive analysis shows that the adsorbent G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ based on G@mSiO ₂ and ligand PFIL = diethyl (3-bromopropyl) phosphate shows better specificity, The reason may be that due to the large specific surface area of mesoporous silica, more hydrophilic polyethylenimine can be modified, and more phosphorous groups can be modified to immobilize more metal ions, thus enriching to more phosphorylated peptides.

(2) In order to study the effect of different metal ions on the enrichment of phosphorylated peptides, we fixed different metal ions (Ga ³⁺ , Ti ⁴⁺ ) on the substrate G@mSiO ₂ , the ligand PFIL=diethyl(3- On bromopropyl) phosphate, the enrichment effect of G@mSiO ₂ @PEI-PFIL-M ⁿ⁺ (Ga ³⁺ , Ti ⁴⁺ ), which immobilized different metal ions, on phosphorylated peptides in β-casein hydrolyzate was compared .

Weigh 2 mg of the two adsorbents G@mSiO ₂ @PEI-PFIL-M ⁿ⁺ (Ga ³⁺ , Ti ⁴⁺ ) in 0.8 mL of enrichment buffer (50% ACN, 0.1% TFA, v/v) , after ultrasonic dispersion, 100 μL of the dispersion liquid was taken out for the enrichment experiment, and 1 μL of standard peptide enzymatic hydrolysis solution (200 fmol/μL) was added to the dispersion liquid. Then, the mixture was placed in a constant temperature metal bath and shaken for 30 min at 37°C. After the reaction was complete, the solid was separated by centrifugation and the solid material was washed three times with enrichment buffer. Finally, disperse the washed solid material with 10 μL of 0.4M ammonia water, shake at 37°C for 15 min, take 5 μL of supernatant after centrifugation, and mix with 5 μL of matrix solution (saturated DHB solution, containing 50% ACN and 0.1% TFA) Finally, 1 μL of the mixed solution was dropped on a MALDI target plate, dried in air, and analyzed by MALDI-TOF MS.

The comparative results of mass spectrometry analysis are shown in Fig. 3 and Fig. 2j. After the samples were treated with two kinds of adsorbents G@mSiO ₂ @PEI-PFIL-M ⁿ⁺ (Ga ³⁺ , Ti ⁴⁺ ), it can be observed that a phosphorylated peptide signal peak was detected in Figure 3, and the background baseline was relative to The material G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (Fig. 2j) is higher, and after the sample is treated with G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (Fig. 2j), 5 phosphorylated peptides can be detected Signal peaks, from the spectrum background and the number of detected phosphorylated peptide signal peaks, the material G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ can better enrich polyphosphorylated peptides.

(3) In order to further study the effect of different metal ions on the specific enrichment of phosphorylated peptides, the material G@mSiO ₂ @PEI-PFIL-M ⁿ⁺ (Ga ³⁺ , Ti ⁴⁺ ) immobilized with different metal ions was compared to the Enrichment effect of phosphorylated peptides in β-casein and bovine serum albumin-BSA enzymatic hydrolysis mixture (molar ratio 1:5000).

Dissolve 1mg bovine serum albumin in 0.1mL 50mM ammonium bicarbonate denaturing buffer (including 8M urea), add 0.2mL 0.1M dithiothreitol (DTT) solution after denaturation, and react at 37°C for 30min to reduce the protein Then add 0.2mL 0.2M iodoacetamide (IAA) solution, and react in the dark for 30min at room temperature to alkylate the reduced mercapto groups; =8.3) was diluted to 1 mL; trypsin was added to the mixed solution (the mass ratio of trypsin to substrate was 1:50), and reacted at 37°C for 16h. The product after enzymatic hydrolysis was stored in a refrigerator at -20°C until use.

Weigh 2 mg of the two adsorbents G@mSiO ₂ @PEI-PFIL-M ⁿ⁺ (Ga ³⁺ , Ti ⁴⁺ ) into 1 mL of enrichment buffer (50% ACN, 0.1% TFA, v/v), After ultrasonic dispersion, 100 μL of the dispersion liquid was taken out for the enrichment experiment, and 100 μL of proteolysis mixture was added to the dispersion liquid. Then, the mixture was placed in a constant temperature metal bath, shaken at 37° C. for 30 min, the solid was separated by centrifugation, and the solid material was washed three times with enrichment buffer. After the reaction, disperse the washed solid material with 10 μL of 0.4M ammonia water, shake it at 37°C for 15 minutes, take 5 μL of the supernatant after centrifugation, and mix with 5 μL of matrix solution (saturated DHB solution, containing 50% ACN and 0.1% TFA ) after mixing, 1 μL of the mixed solution was dropped on a MALDI target plate, dried in the air, and analyzed by MALDI-TOF MS.

The analysis results are shown in Figure 4 series. Figure 4a shows that after G@mSiO ₂ @PEI-PFIL-Ga ³⁺ treated samples, five phosphorylated peptide signal peaks could be detected, but there were some non-phosphorylated peptide signals, and the baseline Higher; Figure 4b shows that after the sample was treated with G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ , the signals of 5 phosphorylated peptides and 1 dephosphorylated residue could be observed, and there were less non-phosphorylated peptides, background relatively clean. Therefore, from the comprehensive analysis of the enrichment results of the two enriched samples, G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ has better specificity for the enrichment of phosphorylated peptides.

(4) In order to better evaluate the adsorption capacity of G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ nanocomposites to phosphorylated peptides, we chose more complex proteolysis solutions as adsorption samples, that is, to continue to increase the concentration of β-casein and the molar ratio of the BSA enzymolysis solution in the bovine serum albumin BSA enzymolysis mixture (the molar ratio is 1:12000).

Weigh 2 mg of G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ into 0.8 mL of enrichment buffer (50% ACN, 1% TFA, v/v), after ultrasonic dispersion, take out 100 μL of the dispersion for enrichment To collect experiments, add 240 μL of proteolysis mixture (wherein, the content of β-casein is 1.43 pmol) to the dispersion. Then, the mixture was placed in a constant temperature metal bath, shaken at 37° C. for 30 min, the solid was separated by centrifugation, and the solid material was washed three times with enrichment buffer. Finally, disperse the washed solid material with 10 μL of 0.4M ammonia water, shake at 37°C for 15 min, take 5 μL of supernatant after centrifugation, and mix with 5 μL of matrix solution (saturated DHB solution, containing 50% ACN and 0.1% TFA) Finally, 1 μL of the mixed solution was dropped on a MALDI target plate, dried in air, and analyzed by MALDI-TOF MS.

The mass spectrometry results are shown in Figure 5. After G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ enrichment, the baseline can be found to be slightly higher in the spectrum, but 6 phosphorylated peptides can still be observed signal, and the phosphorylated peptide signal dominates the entire mass spectrum, and the relative intensity of the phosphorylated peptide is high, so the material G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ has good specificity for the enrichment of the phosphorylated peptide.

(5) Enrichment of endogenous phosphorylated peptides in saliva by three enrichment materials A@PEI-PFIL-Ti ⁴⁺ (A=nSiO ₂ , Fe ₃ O ₄ @nSiO ₂ and G@mSiO ₂ ) Set; PFIL = diethyl(3-bromopropyl) phosphate.

Weigh 2mg of the three adsorbents nSiO ₂ @PEI-PFIL-Ti ⁴⁺ , Fe ₃ O ₄ @nSiO ₂ @PEI-PFIL-Ti ⁴⁺ and G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ in 0.8mL In the enrichment buffer (50% ACN, 0.1% TFA, v/v), after ultrasonic dispersion, 100 μL of the dispersion was taken out for the enrichment experiment, and 20 μL of the saliva sample was added to the dispersion. Then, the mixture was placed in a constant temperature metal bath and shaken for 30 min at 37°C. After the reaction was complete, the solid was separated by centrifugation and the solid material was washed three times with enrichment buffer. Finally, disperse the washed solid material with 10 μL of 0.4M ammonia water, shake at 37°C for 15 min, take 5 μL of supernatant after centrifugation, and mix with 5 μL of matrix solution (saturated DHB solution, containing 50% ACN and 0.1% TFA) Finally, 1 μL of the mixed solution was dropped on a MALDI target plate, dried in air, and analyzed by MALDI-TOF MS.

The analysis results are shown in Figure 6 series. Figure 6a is the mass spectrum of the saliva sample directly analyzed by mass spectrometry. It can be observed from the figure that the non-phosphorylated peptide and impurity signal peaks dominate the entire spectrum; nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (Fig. 6b), Fe ₃ O ₄ @nSiO ₂ @PEI-PFIL-Ti ⁴⁺ (Fig. 6c) and G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ (Fig. 6d) for sample treatment Finally, the number of signal peaks of phosphorylated peptides that could be detected were 25, 16 and 27, indicating that these three different substrate materials could be used to enrich endogenous phosphorylated peptides in saliva. From the results of mass spectrometry analysis, it can be seen that relatively few endogenous phosphorylated peptides are enriched in Figure 6b, and the signal of non-phosphorylated peptides is strong, and a large number of phosphorylated peptides are enriched in Figure 6c and 6d, but The phosphorylated peptides enriched in 6d are more clearly displayed in the mass spectrum. The above analysis results show that the three kinds of adsorbents with different substrates have good affinity for the enrichment of phosphorylated peptides, and G@ mSiO ₂ @PEI-PFIL-Ti ⁴⁺ had the best specificity for the enrichment of endogenous phosphorylated peptides in saliva.

(6) In order to demonstrate the size exclusion effect of mesoporous silica based on material design, we used the material G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ to treat β-casein hydrolyzate (β-casein), β- Casein (β-casein protein) and bovine serum albumin (BSAprotein) were enriched in a certain mass ratio.

Weigh 2 mg of adsorbent G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ in 0.8 mL of enrichment buffer (50% ACN, 0.1% TFA, v/v), after ultrasonic dispersion, take out 100 μL of the dispersion for use In the enrichment experiment, 10.1 μL and 19.2 μL of β-casein hydrolyzate (β-casein), β-casein protein (β-caseinprotein) and bovine serum albumin (BSAprotein) mixed solution were added to the dispersion respectively. Then, the mixture was placed in a constant temperature metal bath and shaken for 30 min at 37°C. After the reaction was complete, the solid was separated by centrifugation and the solid material was washed three times with enrichment buffer. Finally, disperse the washed solid material with 10 μL of 0.4M ammonia water, shake at 37°C for 15 min, take 5 μL of supernatant after centrifugation, and mix with 5 μL of matrix solution (saturated DHB solution, containing 50% ACN and 0.1% TFA) Finally, take 1 μL of the mixed solution and drop it on the MALDI target plate, dry it in the air and perform MALDI-TOF MS analysis;

The test results are shown in Figure 7 series, where Figures 7a and 7b show the effects of the material G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ on β-casein hydrolyzate (β-casein): β-casein (β- caseinprotein): bovine serum albumin (BSAprotein) β-casein hydrolysis = 1:1000:1000, β-casein hydrolysis (β-casein): β-casein (β-caseinprotein): bovine serum albumin (BSAprotein) β-casein hydrolysis = 1:2000:2000 detection results, it can be seen from the figure that when the mass ratio in Figure 7a is 1:1000:1000, 4 signal peaks of phosphorylated peptides and 2 dephosphorylated peptides can be detected Residue signal peaks, and the peak signal has a large intensity. When the mass ratio in Figure 7b is 1:2000:2000, it can still be detected that the number of phosphorylated peptide signal peaks has not decreased, and the background of the spectrum is relatively clean, and the phosphorylated peptide peak is dominant. From the analysis of the results, it can be concluded that the adsorption Agent G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ can block a large number of large molecular weight proteins, has a good protein size exclusion effect, and has great potential in the study of endogenous phosphorylated peptides.

(7) In order to demonstrate the stability and reusability of material design, we used the material G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ to enrich β-casein hydrolyzate 10 times.

Weigh 2 mg of adsorbent G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ in 800 μL of enrichment buffer (50% ACN, 0.1% TFA, v/v), after ultrasonic dispersion, take out 100 μL of the dispersion for For enrichment experiments, β-casein hydrolyzate (200 fmol) was added to the dispersion. Then, the mixture was placed in a constant temperature metal bath, shaken at 37° C. for 30 min, the solid was separated by centrifugation, and the solid material was washed three times with enrichment buffer. Finally, disperse the washed solid material with 10 μL of 0.4M ammonia water, shake at 37°C for 15 min, take 5 μL of supernatant after centrifugation, and mix with 5 μL of matrix solution (saturated DHB solution, containing 50% ACN and 0.1% TFA) Finally, take 1 μL of the mixed solution and drop it on the MALDI target plate, dry it in the air and perform MALDI-TOFMS analysis; the desorbed material is washed three times with the enrichment buffer, and then the next enrichment is carried out under the same conditions. Set-desorption cycle experiments.

The detection results are shown in Figure 8 series, where Figures 8a and 8b are the enrichment results of the material G@mSiO ₂ @PEI-PFIL-Ti ⁴⁺ on the β-casein hydrolyzate after the 1st and 5th repeated use respectively. As can be seen from the figure, the enrichment effects of the 1st and 5th times are similar; compared with the 1st and 5th test results, after 10 times of repeated enrichment (Figure 8c), in In the mass spectrum, 4 phosphorylated peptide peak signals can be observed, and there is not much difference in the number of detected phosphorylated peptides. The above results show that the material still maintains good adsorption capacity after multiple enrichment, showing good stability and reusability.

The scope of protection of the present invention includes but is not limited to the above embodiments. The scope of protection of the present invention is based on the claims. Any replacement, deformation, and improvement that are easily conceived by those skilled in the art for this technology fall within the scope of the present invention. protected range.

Claims (7)

1. The phosphine-based ionic liquid modified nanocomposite is characterized in that the material is prepared by the following method:

(1) Dispersing the substrate material A in anhydrous toluene, adding 3-chloropropyl triethoxysilane, and adding N ₂ Continuously 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 any one of nano silicon dioxide, magnetic core-shell structure nano silicon dioxide or mesoporous silicon dioxide coated graphene;

(2) Dissolving PEI in absolute ethyl alcohol, uniformly stirring, adding the obtained material A-CP into the solution, stirring, heating, and grafting PEI on the surface of the obtained A-CP to obtain material A@PEI;

(3) Dispersing the obtained material A@PEI in anhydrous toluene, adding excessive bromoalkyl phosphate into the material, stirring, heating for reaction, and then washing and drying to obtain a phosphino functionalized ionic liquid modified material, namely A@PEI-PFIL; the brominated alkyl phosphate 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 into concentrated hydrobromic acid which is 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 2 hours at 37 ℃ to obtain an affinity material for fixing metal ions, washing and drying to obtain the nano material A@PEI-PFIL-M ⁿ⁺ Wherein M is ⁿ⁺ ＝Ti ⁴⁺ ,Ga ³⁺ The method comprises the steps of carrying out a first treatment on the surface of the The metal salt solution is Ti (SO) ₄ ) ₂ Or GaCl ₃ 。

2. The preparation method of the phosphine-based ionic liquid modified nanocomposite is characterized by comprising the following steps of:

(1) Dispersing the substrate material A in anhydrous toluene, adding 3-chloropropyl triethoxysilane, and adding N ₂ Continuously stirring and heating under the atmosphere to obtain the chloropropyl modified nano material, namely A-CP, washing and drying; the substrate material A isAny one of nano silicon dioxide, magnetic core-shell structure nano silicon dioxide or mesoporous silicon dioxide coated graphene;

(2) Dissolving PEI in absolute ethyl alcohol, uniformly stirring, adding the obtained material A-CP into the solution, stirring, heating, and grafting PEI on the surface of the obtained A-CP to obtain material A@PEI;

(3) Dispersing the obtained material A@PEI in anhydrous toluene, adding excessive bromoalkyl phosphate into the material, stirring, heating for reaction, washing and drying to obtain a phosphino-functionalized ionic liquid modified material, namely A@PEI-PFIL; the brominated alkyl phosphate 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 into concentrated hydrobromic acid which is 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 2 hours at 37 ℃ to obtain an affinity material for fixing metal ions, washing and drying to obtain the nano material A@PEI-PFIL-M ⁿ⁺ Wherein M is ⁿ⁺ ＝Ti ⁴⁺ ,Ga ³⁺ The method comprises the steps of carrying out a first treatment on the surface of the The metal salt solution is Ti (SO) ₄ ) ₂ Or GaCl ₃ 。

3. The method for preparing a phosphine-based ionic liquid modified nanocomposite according to claim 2, wherein in the step (1), the reaction temperature is 85 ℃ and the reaction time is 14h.

4. The method for preparing a phosphine-based ionic liquid modified nanocomposite according to claim 2, wherein in the step (2), the reaction temperature is 80 ℃ and the reaction time is 24 hours.

5. The method for preparing a phosphine-based ionic liquid modified nanocomposite according to claim 2, wherein in the step (3), the reaction temperature is 110 ℃ and the reaction time is 16h.

6. The method for preparing a phosphine-based ionic liquid modified nanocomposite according to claim 2, wherein the detergents in steps (1), (2) and (3) are ethanol.

7. Use of a phosphine based ionic liquid modified nanocomposite for enriching phosphorylated peptides, characterized in that the phosphine based ionic liquid modified nanocomposite of claim 1 is used for enriching phosphorylated peptides.

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