CN114272915B - Phosphine-based ionic liquid modified nanocomposite, preparation method thereof and application thereof in enrichment of phosphorylated peptides - Google Patents
Phosphine-based ionic liquid modified nanocomposite, preparation method thereof and application thereof in enrichment of phosphorylated peptides Download PDFInfo
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Abstract
A phosphino ionic liquid modified nano-composite material and a preparation method thereof are provided, and the material is applied to enrichment of phosphorylated peptides in biological samples containing the phosphorylated peptides. The method comprises the following steps: (1) The method comprises the steps of (1) fixing a base material containing chloropropyl modification on the surface of the base material through substitution reaction with Polyethyleneimine (PEI); (2) And carrying out quaternization reaction on the brominated alkyl phosphonate and the PEI layer to obtain the ionic liquid modified layer with organic phosphine functionalization. (3) And fixing metal ions on the surface of the material through ester group hydrolysis reaction and metal ion complexation reaction. The functional material prepared by the invention has larger specific surface area, excellent hydrophilicity, more metal immobilization capacity, excellent sensitivity, high specific selectivity and size exclusion effect and excellent reusability. The nanocomposite is suitable for enrichment and purification of phosphorylated peptides of complex biological samples. The material has wide application prospect in the field of proteomics and biomedicine.
Description
Technical Field
The invention belongs to the field of functional materials and life science, in particular relates to a phosphine-based ionic liquid modified nanocomposite, and also relates to a preparation method of the material and application of the material in enrichment of phosphorylated peptides.
Background
In the 90 s of the 20 th century, with the rapid development of genomics, human genome sequencing work was essentially complete. It is becoming appreciated that studies based on mRNA levels cannot contain all information about life, and thus direct executors of vital activities-protein studies are becoming a hotspot. It is counted that more than 30% of the proteins are phosphorylated to varying degrees in mammalian cells. Protein phosphorylation is the most widespread post-translational modification in nature, and different post-translational modifications play different roles in the regulation of vital activities, such as 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 been widely used in the field of proteomics research. In complex biological samples, large amounts of non-phosphorylated peptides are present, however, the amount of phosphorylated proteins is very small and ionization efficiency in mass spectrometry is low, which results in the inhibition of low abundance phosphorylated peptide signals. Therefore, designing an enrichment material that efficiently captures the phosphorylated peptides prior to mass spectrometry is critical for accurate identification of phosphorylated peptide information.
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 have been synthesized to capture the phosphorylated peptide or the phosphorylated protein according to different mechanisms of the phosphorylated peptide enrichment, and the traditional methods solve the enrichment problem of the phosphorylated peptide, but some problems such as weak anti-interference, low detection sensitivity, low selectivity, poor capability of eliminating biological macromolecule interference and the like can not be well avoided.
Disclosure of Invention
Based on the problems existing in the prior art, the invention introduces a new precursor Polyethylenimine (PEI), improves the nitrogen content of the surface of the material, and obtains the organic phosphorus functional ionic liquid with high specific content through quaternization and cross-linking of halogenated alkyl organic phosphate and PEI, thereby being beneficial to improving the metal ion content and further improving the enrichment capacity, sensitivity and selectivity of the enrichment material on the phosphorylated peptide.
In order to solve the technical problems, the invention is solved by the following technical scheme.
The phosphine-based ionic liquid modified nanocomposite material is prepared by the following method: (1) Dispersing the base material A in anhydrous toluene, adding 3-chloropropyl triethoxysilane, and dispersing in 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 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 ) Any one of them; (2) Dissolving PEI in absolute ethyl alcohol, uniformly stirring, 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) Dispersing the obtained material A@PEI in anhydrous toluene, adding excessive bromoalkyl phosphate into the anhydrous toluene, stirring and heating the mixture to react. Washing and drying to obtain the phosphino functionalized ionic liquid modified material, namely A@PEI-PFIL; the bromoalkyl phosphate is any one of the following 3 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 2 times by deionized water, stirring, heating, neutralizing with NaOH solution (PH=11), 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+ (M 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 。
The preparation method of the phosphine-based ionic liquid modified nanocomposite comprises the following steps:
(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 nanometerThe material, namely A-CP, is washed and dried; the substrate 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 ) Any one of them. (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 a material A@PEI; (3) Dispersing the obtained material A@PEI in anhydrous toluene, adding excessive bromoalkyl phosphate into the anhydrous toluene, stirring and heating the mixture to react. Washing and drying to obtain the phosphino functionalized ionic liquid modified material, namely A@PEI-PFIL; the bromoalkyl phosphate is any one of the following 3 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 2 times by deionized water, stirring, heating, neutralizing with NaOH solution (PH=11), 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+ (M 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 。
Further, in the step (1), the reaction temperature is 85 ℃ and the reaction time is 14h.
Further, in the step (2), the reaction temperature is 80 ℃ and the reaction time is 24 hours.
Further, in the step (3), the reaction temperature is 110 ℃, and the reaction time is 16h.
Further, the washing solutions in the steps (1), (2) and (3) are all ethanol.
The application of the phosphine-based ionic liquid modified nanocomposite in the enrichment of the phosphorylated peptides comprises the step of using the phosphine-based ionic liquid modified nanocomposite in the enrichment of the phosphorylated peptides.
The principle of the invention is as follows: the invention uses bromoalkyl phosphateQuaternizing the polyethyleneimine modified nanomaterial, crosslinking the organophosphorus functional ionic liquid, and modifying the organophosphorus functional ionic liquid on a substrate material to obtain the polyethyleneimine modified nanomaterial, namely A@PEI-PFIL; after the acidification treatment, metal ions (M) n+ ) Obtaining A@PEI-PFIL-M n+ Fixing metal ion affinity chromatographic material.
The invention is characterized in that the combination of the base material A and the bromoalkyl phosphate is changed, namely: (1) selecting nano silicon dioxide, silicon dioxide coated magnetic sphere nano particles and mesoporous silicon dioxide coated graphene and diethyl (3-bromopropyl) phosphate nano composite material, (2) nano silicon dioxide, silicon dioxide coated magnetic sphere nano particles and mesoporous silicon dioxide coated graphene and diethyl (4-bromobutyl) phosphate nano composite material, (3) nano silicon dioxide, silicon dioxide coated magnetic sphere nano particles and mesoporous silicon dioxide coated graphene and diethyl (5-bromopentyl) phosphate nano composite material, and preparing nine IMAC adsorbents with different substrates and different ligand materials, namely 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+ )。
Compared with the prior art, the invention has the following beneficial effects: (1) The modification process is simple, easy to operate, and the shape of the substrate material is not changed, and the prepared material has good environmental stability and reusability. (2) The immobilized metal ion affinity material synthesized in the invention: A@PEI-PFIL-M n+ (M n+ ＝Ti 4+ ,Ga 3+ ) The IMAC adsorption material utilizes the affinity of the phosphorylated peptide and the immobilized metal ion, can be used for the specific enrichment of the phosphorylated peptide, and the synthesized material effectively solves the problems of insufficient enrichment selectivity, low adsorption specific capacity, poor protein exclusion effect and the like, and can be used for the enrichment of the phosphorylated peptide in complex biological samples.
Drawings
FIG. 1 is a nanocomposite A@PEI-PFIL-M n+ (wherein a=nsio 2 、Fe 3 O 4 @nSiO 2 Or G@mSiO 2 The method comprises the steps of carrying out a first treatment on the surface of the PFIL = diethyl (3-bromopropyl) phosphate, diethyl (4-bromobutyl) phosphate or diethyl (5-bromopentyl) phosphate, M n+ ＝Ti 4+ ,Ga 3+ ) A synthetic flow chart.
FIGS. 2 a-2 j are mass spectra of beta-casein enzymatic hydrolysate (200 fmol/. Mu.L); phosphorylated peptide signal is represented by x, dephosphorylated residue is represented by # wherein:
FIG. 2a is a direct assay of beta-casein enzymatic hydrolysate;
FIG. 2b shows the beta-casein enzymatic hydrolysate subjected to nSiO 2 @PEI-PFIL-Ti 4+ (PFIL = diethyl (5-bromopentyl) phosphate) post-treatment mass spectrum;
FIG. 2c shows the beta-casein enzymatic hydrolysate subjected to nSiO 2 @PEI-PFIL-Ti 4+ Mass spectrum after (PFIL = diethyl (4-bromobutyl) phosphate treatment;
FIG. 2d shows the beta-casein enzymatic hydrolysate subjected to nSiO 2 @PEI-PFIL-Ti 4+ Mass spectrum after (PFIL = diethyl (3-bromopropyl) phosphate treatment;
FIG. 2e shows the beta-casein enzymatic hydrolysate after Fe3O4@nSiO 2 @PEI-PFIL-Ti 4+ (PFIL = diethyl (5-bromopentyl) phosphate) post-treatment mass spectrum;
FIG. 2f shows the beta-casein enzymatic hydrolysate after Fe3O4@nSiO 2 @PEI-PFIL-Ti 4+ Mass spectrum after (PFIL = diethyl (4-bromobutyl) phosphate treatment;
FIG. 2g shows the beta-casein enzymatic hydrolysate after Fe3O4@nSiO 2 @PEI-PFIL-Ti 4+ Mass spectrum after (PFIL = diethyl (3-bromopropyl) phosphate treatment;
FIG. 2h shows the beta-casein enzymatic hydrolysate subjected to G@mSiO 2 @PEI-PFIL-Ti 4+ (PFIL = diethyl (5-bromopentyl) phosphate) post-treatment mass spectrum;
FIG. 2i shows the beta-casein enzymatic hydrolysate after G@mSiO 2 @PEI-PFIL-Ti 4+ Mass spectrum after (PFIL = diethyl (4-bromobutyl) phosphate treatment;
FIG. 2j shows beta-casein enzymatic hydrolysateThrough G@mSiO 2 @PEI-PFIL-Ti 4+ (PFIL = diethyl (3-bromopropyl) phosphate).
FIG. 3 shows the reaction of beta-casein enzymatic hydrolysate with G@mSiO 2 @PEI-PFIL-Ga 3+ A mass spectrogram after treatment; PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signal is represented by x, dephosphorylated residue is represented by # and p.
FIGS. 4 a-4 b are mass spectra of enzymatic hydrolysis mixtures of beta-casein and bovine serum albumin BSA (molar ratio 1:5000); PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signal is represented by x, dephosphorylated residue is represented by #; wherein:
FIG. 4a is G@mSiO 2 @PEI-PFIL-Ga 3+ A mass spectrogram after treatment;
FIG. 4b is G@mSiO 2 @PEI-PFIL-Ti 4+ And (5) a mass spectrogram after treatment.
FIG. 5 shows the enzymatic hydrolysis mixture of beta-casein (1.43 pmol) and bovine serum albumin BSA (molar ratio 1:12000) at G@mSiO 2 @PEI-PFIL-Ti 4+ A mass spectrogram after treatment; PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signal is represented by x, dephosphorylated residue is represented by # and p.
FIGS. 6 a-6 d are mass spectra of saliva; PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signal is represented by x and dotted or conventional numbers; wherein:
FIG. 6a is a mass spectrum of a saliva sample for direct analysis;
FIG. 6b is nSiO 2 @PEI-PFIL-Ti 4+ A mass spectrogram after treatment;
FIG. 6c is Fe 3 O 4 @nSiO 2 @PEI-PFIL-Ti 4+ A mass spectrogram after treatment;
FIG. 6d is G@mSiO 2 @PEI-PFIL-Ti 4+ And (5) a mass spectrogram after treatment.
FIGS. 7 a-7 b are adsorbents G@mSiO 2 @PEI-PFIL-Ti 4+ A mass spectrum of protein exclusion; PFIL = diethyl (3-bromopropyl) phosphate, phosphorylated peptide signal is represented by x, dephosphorylated residue is represented by #; wherein:
FIG. 7a is beta-casein protein: bsapretin=1:1000:1000 mass spectrum;
fig. 7b is a mass spectrum of β -casein: β -casein protein: bsapretin=1:2000:2000.
FIGS. 8 a-8 c are mass spectra of beta-casein enzymatic hydrolysate; phosphorylated peptide signal is represented by x, dephosphorylated residue is represented by #; wherein:
FIG. 8a is G@mSiO 2 @PEI-PFIL-Ti 4+ A mass spectrum chart of the 1 st enrichment of the beta-casein enzymatic hydrolysate;
FIG. 8b is G@mSiO 2 @PEI-PFIL-Ti 4+ Repeatedly using the beta-casein enzymolysis liquid for 5 times to enrich mass spectrograms;
FIG. 8c is G@mSiO 2 @PEI-PFIL-Ti 4+ And (5) repeatedly using the beta-casein enzymolysis liquid for 10 times to enrich the mass spectrum.
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
In the following embodiments, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout, and the embodiments described below by referring to the drawings are exemplary only for explaining the present invention and are not to be construed as limiting the present invention.
Example 1: wherein a=nsio 2 PFIL = diethyl (5-bromopentyl) phosphate.
Example 2: wherein a=nsio 2 PFIL = diethyl (4-bromobutyl) phosphate.
Example 3: wherein a=nsio 2 PFIL = diethyl (3-bromopropyl) phosphate.
Example 4: wherein a=fe 3 O 4 @nSiO 2 PFIL = diethyl (5-bromopentyl) phosphate.
Example 5: wherein a=fe 3 O 4 @nSiO 2 PFIL = diethyl (4-bromobutyl) phosphate.
Example 6: wherein a=fe 3 O 4 @nSiO 2 PFIL = diethyl (3-bromopropyl) phosphate.
Example 7: wherein a=g@msio 2 PFIL = diethyl (5-bromopentyl) phosphate.
Example 8: wherein a=g@msio 2 PFIL = diethyl (4-bromobutyl) phosphate.
Example 9: wherein a=g@msio 2 PFIL = diethyl (3-bromopropyl) phosphate.
In the above 9 embodiments, the difference is that the substrate material a and the ligand PFIL are different, and the preparation method is the same: firstly preparing a chloropropyl modified nano-composite to obtain an A-CP, grafting polyethyleneimine on the surface of the prepared nano-composite, and then carrying out cross-linking quaternization by using bromoalkyl phosphate and polyethyleneimine, and modifying the surface of the nano-composite to obtain the A@PEI-PFIL; after acidification treatment, metal ions are fixed on the organic phosphate group, thus obtaining the fixed metal ion affinity adsorbent A@PEI-PFIL-M n+ 。
The preparation method comprises the following steps:
(1) The preparation method of the chloropropyl modified nano material comprises the following steps: 200mg of the base material was dispersed in 30mL of anhydrous toluene, after ultrasonic treatment was performed for 10 minutes, 0.6mL of 3-chloropropyl triethoxysilane was added thereto, and the mixture was heated to 85℃under nitrogen atmosphere, and reacted with stirring for 14 hours. After the reaction is finished, centrifugally separating the solid, washing the solid with ethanol for several times, and drying the solid to obtain the A-CP.
(2) Preparation of A@PEI: 1g of polyethyleneimine was dissolved in 25mL of absolute ethanol, and after stirring for 10 minutes, 200mg of A-CP was dispersed in the polyethyleneimine absolute ethanol solution, followed by ultrasonic treatment for 10 minutes, heating to 80℃and stirring for reaction for 24 hours. After the reaction is finished, centrifugally separating solids, washing the solids with deionized water and ethanol for several times in sequence, and drying the solids to obtain the A@PEI.
(3) Preparation of A@PEI-PFIL: 0.5g of bromoalkylphosphate is added dropwise to 15mL of anhydrous toluene and sonicated for ten minutes, 100mg of A@PEI is dispersed in the solution of bromoalkylphosphate in anhydrous toluene and sonicated for 10 minutes and magnetically stirred in an oil bath at 110℃for 16 hours. After the reaction is finished, centrifugally separating the solid, washing the solid with ethanol for several times, and drying the solid to obtain the A@PEI-PFIL.
(4) Acidizing 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 2h. After the reaction is finished, neutralizing with sodium hydroxide solution (pH=11), washing with deionized water to neutrality, and drying to obtain sodium salt A@PEI-PFIL-Na + 。
(5)A@PEI-PFIL-M n+ Is prepared from the following steps: acid-treated A@PEI-PFIL-Na + Dispersed in 15ml of 0.1M metal salt solution and shaken for 2h at room temperature. After the reaction is finished, centrifugally separating solids, washing the solids with deionized water and ethanol in sequence, and drying the solids at 85 ℃ to obtain the A@PEI-PFIL-M n+ . The metal salt solution is Ti (SO) 4 ) 2 Or GaCl 3 A solution.
The experimental test and the description of the drawings are as follows:
(1) To examine the nano materials of 3 different substrates and 3 different ligands in modifying titanium ions (A@PEI-PFIL-Ti) 4+ ) Effect of the subsequent enrichment on phosphorylated peptides, thus determining the effect of different substrate materials on enrichment, we compared nine A@PEI-PFIL-Ti 4+ The enrichment effect of the adsorbent on the phosphorylated peptides in the standard protein beta-casein enzymatic hydrolysate.
5mg of beta-casein was dissolved in 1ml of 25mM ammonium bicarbonate buffer solution (pH=8); trypsin (trypsin to substrate mass ratio 1:50) was added to the mixed solution and reacted at 37℃for 12h. And storing the product after enzymolysis in a refrigerator at the temperature of minus 20 ℃ for standby.
To compare IMAC adsorbents A@PEI-PFIL-Ti of nine different substrates 4+ Enrichment effect on phosphorylated peptides we first selected the standard protein β -casein as the enriched sample.
2mg of each of the 3 different substrates and 3 different ligand adsorbents were weighed into 0.8mL of enrichment buffer (50% ACN,0.1% TFA, v/v), sonicated for 10 min, and 100. Mu.L of the dispersion was removed for enrichment experiments, and 1. Mu.L of standard peptidase solution (200 fmol/. Mu.L) was added to 100. Mu.L of the dispersion. Then, the mixed solution is placed in a constant temperature metal bath, and is vibrated for 30min at 37 ℃, and the solid material is washed three times by centrifugation or magnetic separation of the solid and 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 MALDI target plate, and the mixture was dried in air and analyzed by MALDI-TOF MS.
The mass spectrum detection results are shown in the series of fig. 2, fig. 2a shows the result of mass spectrum analysis directly performed on a sample without treatment, and the signal of the phosphorylated peptide is almost absent from fig. 2 a; after the sample is treated by nine adsorbents, the analysis results are shown as b-j in FIG. 2, and the sample is subjected to 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 4, 3, 5, 2, 4, 3, 5 and 5 phosphorylated peptide signal peaks, respectively, and corresponding 1, 0, 1, 2 and 2 dephosphorylated residue signal peaks, can be detected. According to the mass spectrum result, the signal peak intensity of the phosphorylated peptide is larger, the background is cleaner, and the experiment result shows that all the nine adsorbents can effectively enrich the phosphorylated peptide; from the comprehensive analysis of the number of phosphorylated peptide signals and the condition of the hetero peak, G@mSiO is adopted 2 Adsorbent g@msio for substrate, ligand pfil=diethyl (3-bromopropyl) phosphate 2 @PEI-PFIL-Ti 4+ Exhibits better specificity, probably due toBecause the specific surface area of the mesoporous silica is large, more hydrophilic polyethyleneimine can be modified, more phosphorus oxygen groups are modified to fix more metal ions, and more phosphorylated peptides are enriched.
(2) To study the effect of different metal ions on enrichment of phosphorylated peptides, we performed a reaction between different metal ions (Ga 3+ 、Ti 4+ ) Fixed on a substrate G@mSiO 2 On ligand PFIL=diethyl (3-bromopropyl) phosphate, material G@mSiO for fixing different metal ions is compared 2 @PEI-PFIL-M n+ (Ga 3+ 、Ti 4+ ) The enrichment effect on the phosphorylated peptide in the beta-casein enzymatic hydrolysate.
2mg of two adsorbents G@mSiO are respectively weighed 2 @PEI-PFIL-M n+ (Ga 3+ 、Ti 4+ ) After ultrasonic dispersion 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 1. Mu.L of standard peptidase 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 30min. After the reaction was completed, the solids were 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 to obtain 5. Mu.L of supernatant, mixed with 5. Mu.L of matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. Mu.L of the mixed solution was dropped onto a MALDI target plate, and dried in air for MALDI-TOF MS analysis.
The comparison results of the mass spectrometry are shown in fig. 3 and fig. 2 j. The sample is respectively passed through two adsorbents G@mSiO 2 @PEI-PFIL-M n+ (Ga 3+ 、Ti 4+ ) After treatment, it can be observed that 1 phosphorylated peptide signal peak is detected in FIG. 3, and that the background baseline is relative to the material G@mSiO 2 @PEI-PFIL-Ti 4+ (FIG. 2 j) is higher, sample G@mSiO 2 @PEI-PFIL-Ti 4+ (FIG. 2 j) after the treatment, 5 signal peaks of phosphorylated peptides can be detected, and the material G@mSiO can be known from the background of the spectrogram and the number of detected signal peaks of phosphorylated peptides 2 @PEI-PFIL-Ti 4+ The polyphosphorylated peptide can be better enriched.
(3) To further investigate the effect of different metal ions on the specific enrichment of phosphorylated peptides, the materials G@mSiO immobilizing the different metal ions were compared 2 @PEI-PFIL-M n+ (Ga 3+ 、Ti 4+ ) Enrichment effect on phosphorylated peptides in beta-casein and bovine serum albumin BSA enzymolysis mixed solution (molar ratio is 1:5000).
1mg bovine serum albumin is dissolved in 0.1mL 50mM ammonium bicarbonate denaturation buffer (containing 8M urea), after denaturation, 0.2mL 0.1M Dithiothreitol (DTT) solution is added, and the mixture is reacted at 37 ℃ for 30min to reduce disulfide bonds in the protein, then 0.2mL 0.2M Iodoacetamide (IAA) solution is added, and the mixture is reacted at room temperature for 30min in a dark place to alkylate the reduced sulfhydryl; the above product was diluted to 1mL with 50mM ammonium bicarbonate buffer (ph=8.3); trypsin (trypsin to substrate mass ratio 1:50) was added to the mixed solution and reacted at 37℃for 16h. And storing the product after enzymolysis in a refrigerator at the temperature of minus 20 ℃ for standby.
2mg of two adsorbents G@mSiO are respectively weighed 2 @PEI-PFIL-M n+ (Ga 3+ 、Ti 4+ ) After ultrasonic dispersion in 1mL of enrichment buffer (50% ACN,0.1% TFA, v/v), 100. Mu.L of the dispersion was removed for enrichment experiments, and 100. Mu.L of the proteolytic 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 solids and the solid material washed three times with enrichment buffer. After the reaction, the washed solid material was dispersed with 10. Mu.L of 0.4M aqueous ammonia, shaken at 37℃for 15 minutes, centrifuged to obtain 5. Mu.L of a supernatant, 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 MALDI target plate, and dried in air for MALDI-TOF MS analysis.
The analysis results are shown in the series of FIG. 4, FIG. 4a is G@mSiO 2 @PEI-PFIL-Ga 3+ After treatment of the sample, 5 phosphorylated peptide signal peaks can be detected, but some non-phosphorylated peptide signal is present and baseline is higher; FIG. 4b is G@mSiO 2 @PEI-PFIL-Ti 4+ After treatment of the samples, 5 phosphorylated peptides and 1 dephosphorylation can be observedThe signal of the acid residues, with less non-phosphorylated peptides, was relatively clean in background. 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 the phosphorylated peptide.
(4) To better evaluate G@mSiO 2 @PEI-PFIL-Ti 4+ The adsorption capacity of the nano-composite to the phosphorylated peptide is that we select more complex proteolytic liquid as adsorption sample, namely, the molar ratio of BSA enzymatic hydrolysate in the enzymatic hydrolysate of beta-casein and bovine serum albumin BSA is continuously improved (molar ratio is 1:12000).
2mg of G@mSiO are weighed 2 @PEI-PFIL-Ti 4+ After ultrasonic dispersion in 0.8mL of enrichment buffer (50% ACN,1% TFA, v/v), 100. Mu.L of the dispersion was removed for enrichment experiments, and 240. Mu.L of a proteolytic mixture (wherein the beta-casein content was 1.43 pmol) 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 solids and the solid material 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 to obtain 5. Mu.L of supernatant, mixed with 5. Mu.L of matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. Mu.L of the mixed solution was dropped onto a MALDI target plate, and dried in air for MALDI-TOF MS analysis.
The mass spectrum detection result is shown in FIG. 5, and the mass spectrum detection result is shown in the graph of G@mSiO 2 @PEI-PFIL-Ti 4+ After enrichment, a slight increase in baseline was found in the spectra, but 6 phosphorylated peptide signals could still be observed, and the phosphorylated peptide signals dominate the whole mass spectra, and the relative intensity of the phosphorylated peptides was higher, so the material G@mSiO 2 @PEI-PFIL-Ti 4+ Has good specificity for the enrichment of phosphorylated peptides.
(5) Enrichment material A@PEI-PFIL-Ti of 3 different substrates 4+ (A＝nSiO 2 、Fe 3 O 4 @nSiO 2 And G@mSiO 2 ) Enrichment of endogenous phosphorylated peptides in saliva; PFIL = diethyl (3-bromopropyl) phosphate.
Respectively weigh 2mg threeSeed adsorbent nSiO 2 @PEI-PFIL-Ti 4+ 、Fe 3 O 4 @nSiO 2 @PEI-PFIL-Ti 4+ And G@mSiO 2 @PEI-PFIL-Ti 4+ After ultrasonic dispersion 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 30min. After the reaction was completed, the solids were 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 to obtain 5. Mu.L of supernatant, mixed with 5. Mu.L of matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. Mu.L of the mixed solution was dropped onto a MALDI target plate, and dried in air for MALDI-TOF MS analysis.
The analysis results are shown in the series of fig. 6, and fig. 6a is a mass spectrum of saliva samples after being directly subjected to mass spectrometry, and it can be observed from the figure that the non-phosphorylated peptide and impurity signal peaks dominate the whole spectrum; nSiO 2 @PEI-PFIL-Ti 4+ (FIG. 6 b), fe 3 O 4 @nSiO 2 @PEI-PFIL-Ti 4+ (FIG. 6 c) and G@mSiO 2 @PEI-PFIL-Ti 4+ (FIG. 6 d) the number of signal peaks of phosphorylated peptides that could be detected after sample treatment with 3 adsorbents was 25, 16 and 27, respectively, indicating that the material of each of these three different substrates could be used for enrichment of endogenous phosphorylated peptides in saliva. From the mass spectrometry analysis results, FIG. 6b shows that relatively less endogenous phosphorylated peptide is enriched, the signal of non-phosphorylated peptide is stronger, both FIG. 6c and FIG. 6d are enriched for more phosphorylated peptide, but the phosphorylated peptide enriched in FIG. 6d is more clearly shown in the mass spectrogram, the above analysis results show that adsorbents with 3 different substrates all show better affinity for the phosphorylated peptide, and G@mSiO3 2 @PEI-PFIL-Ti 4+ The specificity for 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@mSiO 2 @PEI-PFIL-Ti 4+ For beta-casein enzymatic hydrolysate (beta-ca)Enrichment experiments were performed on the proteins of sein), beta-casein (beta-casein protein) and bovine serum albumin (BSAprotein) in a certain mass ratio.
2mg of adsorbent G@mSiO was weighed 2 @PEI-PFIL-Ti 4+ After ultrasonic dispersion in 0.8mL of enrichment buffer (50% ACN,0.1% TFA, v/v), 100. Mu.L of dispersion was removed for enrichment experiments, and 10.1. Mu.L and 19.2. Mu.L of a mixed solution of beta-casein enzymatic hydrolysate (beta-casein), beta-casein (beta-casein), 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 30min. After the reaction was completed, the solids were 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 ammonia water, shaken at 37℃for 15min, centrifuged to obtain 5. Mu.L of supernatant, mixed with 5. Mu.L of matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. Mu.L of the mixed liquid was dropped onto a MALDI target plate, and the mixture was dried in air for MALDI-TOF MS analysis;
the detection results are shown in the series of FIG. 7, wherein FIGS. 7a and 7b are the materials G@mSiO 2 @PEI-PFIL-Ti 4+ Respectively carrying out the following steps of: beta-casein: bovine serum protein (bsaplotein) β -casein enzyme = 1:1000:1000, beta-casein enzymatic hydrolysate (beta-casein): beta-casein: bovine serum protein (bsaplotein) beta-casein enzymatic hydrolysis = 1:2000:2000, as can be seen from the graph, the mass ratio in fig. 7a is 1:1000: at 1000, 4 signal peaks of phosphorylated peptides and 2 signal peaks of dephosphorylated residues can be detected, and the peak signal has a great intensity. In fig. 7b, the mass ratio is 1:2000:2000, the number of the signal peaks of the phosphorylated peptide can still be detected without reduction, the spectrogram background is cleaner, the phosphorylated peptide peaks are dominant, and the adsorbent G@mSiO can be obtained by analyzing the result 2 @PEI-PFIL-Ti 4+ Can block a large amount of protein with large molecular weight, has good size exclusion effect, and has great potential in researching endogenous phosphorylated peptide.
(7) To demonstrate stability characteristics based on material designAnd reusability, we use the material G@mSiO 2 @PEI-PFIL-Ti 4+ The beta-casein enzymatic hydrolysate is subjected to 10 times of repeated enrichment.
2mg of adsorbent G@mSiO was weighed 2 @PEI-PFIL-Ti 4+ After ultrasonic dispersion in 800. Mu.L of enrichment buffer (50% ACN,0.1% TFA, v/v), 100. Mu.L of the dispersion was removed for enrichment experiments and beta-casein hydrolysate (200 fmol) 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 solids and the solid material washed three times with enrichment buffer. Finally, the washed solid material was dispersed with 10. Mu.L of 0.4M ammonia water, shaken at 37℃for 15min, centrifuged to obtain 5. Mu.L of supernatant, mixed with 5. Mu.L of matrix solution (saturated DHB solution containing 50% ACN and 0.1% TFA), and 1. Mu.L of the mixed liquid was dropped onto a MALDI target plate, and the mixture was dried in air for MALDI-TOF MS analysis; the desorbed material is washed three times with enrichment buffer, and then the next enrichment-desorption cycle experiment is performed under the same conditions.
The detection results are shown in the series of FIG. 8, wherein FIGS. 8a and 8b are the materials G@mSiO 2 @PEI-PFIL-Ti 4+ The detection results of enrichment after the 1 st and 5 th repeated use of the beta-casein enzymatic hydrolysate are respectively shown that the enrichment effect of the 1 st and 5 th is similar; after 10 enrichments with repeated use (fig. 8 c), 4 phosphopeptide peak signals could be observed in the mass spectrum, and no significant difference in the amount of phosphopeptide was detected, compared to the 1 st and 5 th detection results. The results show that the material still maintains good adsorption capacity after multiple enrichment, and has good stability and reusability.
The scope of the present invention includes, but is not limited to, the above embodiments, and any alterations, modifications, and improvements made by those skilled in the art are intended to fall within the scope of the invention.
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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| 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 |
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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 |
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