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 PDF

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CN114272915B
CN114272915B CN202111460502.9A CN202111460502A CN114272915B CN 114272915 B CN114272915 B CN 114272915B CN 202111460502 A CN202111460502 A CN 202111460502A CN 114272915 B CN114272915 B CN 114272915B
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phosphate
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diethyl
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CN114272915A (en
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夏成龙
梁洪泽
王斌斌
蒋雨菲
谢仪珊
周晨阳
李咏梅
赵玲玲
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Ningbo University
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Abstract

一种膦基离子液体修饰纳米复合材料及其制备方法,该材料在含有磷酸化肽的生物样品中,富集磷酸化肽的应用。包括以下步骤:(1)将含有氯丙基修饰的基底材料与聚乙烯亚胺(PEI)通过取代反应,将PEI固定在基底材料表面;(2)将含溴代烷基膦酸酯与PEI层发生季铵化反应,获得具有有机膦功能化的离子液体修饰层。(3)经酯基水解反应、金属离子络合反应,将金属离子固定于材料表面。本发明制备的功能化材料具有较大的比表面积,优越的亲水性,较多的金属固载量,表现出优异的灵敏度,高的特异选择性和尺寸排阻效应以及优良的重复使用性。该类纳米复合材料适用于复杂生物样品的磷酸化肽的富集纯化。该材料在蛋白组学、生物医学领域具有广泛的应用前景。

Figure 202111460502

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.

Figure 202111460502

Description

膦基离子液体修饰纳米复合材料及其制备方法及其在富集磷酸化肽中的应用Phosphine-based ionic liquid modified nanocomposite material and its preparation method and its application in enriching phosphorylated peptides

技术领域technical field

本发明属于功能材料、生命科学领域,具体涉及膦基离子液体修饰纳米复合材料,同时本发明还涉及该材料的制备方法及其在富集磷酸化肽中的应用。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.

背景技术Background technique

20世纪90年代,随着基因组学的快速发展,人类基因组测序工作基本完成。人们开始认识到基于mRNA水平的研究不能包含生命的全部信息,因此对于生命活动的直接执行者-蛋白质研究逐渐成为了热点。据统计,在哺乳动物细胞中,30%以上的蛋白质会发生不同程度的磷酸化。蛋白质磷酸化是大自然中最为广泛的翻译后修饰,且不同的翻译后修饰在生命活动调节中发挥着不同作用,如在信号传导、免疫应答、细胞分化和凋谢等方面起着关键调控作用(Pawson T,Scott J D.Trends Biochem Sci,2005,30(6):286)。对蛋白质磷酸化的全面了解,对疾病诊断学和病理学研究具有十分重要的生物学意义和临床应用价值。近年来,质谱技术得到了快速发展,也已经广泛的应用到了蛋白质组学研究领域中。在复杂的生物样品中,存在着大量的非磷酸化肽,然而磷酸化蛋白的含量却是十分微少的且在质谱中离子化效率低,这就使得低丰度的磷酸化肽信号被抑制。因此,在质谱分析前,设计出一种高效捕获磷酸化肽的富集材料是精确识别磷酸化肽信息的关键。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.

富集磷酸化肽传统的方法有免疫亲和色谱法、金属氧化物亲和色谱法和固定金属离子亲和色谱法等。目前,根据磷酸化肽富集的不同机理已经合成了多种功能性材料来捕获磷酸化肽或磷酸化蛋白,这些传统的方法,解决了磷酸化肽的富集问题,而有一些问题仍然不能很好的避免,如抗干扰弱、检测灵敏度低、选择性低、排除生物大分子干扰能力差等。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.

发明内容Contents of the invention

基于现有技术所存在的问题,本发明引入一种新的前驱体聚乙烯亚胺(PEI),提高材料表面氮含量,再通过卤代烷基有机磷酸酯与PEI季胺化交联,获得高比含量的有机磷功能离子液体,有利于提高金属离子含量,从而提高富集材料对磷酸化肽的富集容量、灵敏度和选择性。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.

为了解决上述技术问题,本发明通过下述技术方案得以解决。In order to solve the above technical problems, the present invention is solved through the following technical solutions.

本申请中的膦基离子液体修饰纳米复合材料,通过以下方法制备:(1)将基底材料A分散在无水甲苯中,加入3-氯丙基三乙氧基硅烷,通过在N2氛围下连续搅拌、加热,得到氯丙基修饰的纳米材料,即A-CP,洗涤、烘干;所述的基底材料A为纳米二氧化硅(nSiO2)、磁性核壳结构纳米二氧化硅(Fe3O4@nSiO2)或介孔二氧化硅包覆的石墨烯(G@mSiO2)中任一种;(2)将PEI溶解在无水乙醇中,搅拌均匀后,向该溶液中加入上述得到的材料A-CP,搅拌、加热后,将得到在A-CP表面接枝一层有聚乙烯亚胺的功能材料,即A@PEI;(3)将得到的材料A@PEI分散在无水甲苯中,然后向其中加入过量溴代烷基磷酸酯,搅拌、加热反应。洗涤、烘干,即可得到膦基功能化离子液体修饰材料,即A@PEI-PFIL;所述的溴代烷基磷酸酯为以下3种任意一种:二乙基(3-溴丙基)磷酸酯、二乙基(4-溴丁基)磷酸酯和二乙基(5-溴戊基)磷酸酯;(4)将得到的材料A@PEI-PFIL分散于去离子水稀释2倍的浓氢溴酸中,搅拌、加热后,用NaOH溶液(PH=11)进行中和,烘干;(5)将步骤(4)得到的材料分散于金属盐溶液中,在37℃下反应2h,便可得到固定金属离子的亲和材料,洗涤、烘干后,即得纳米材料A@PEI-PFIL-Mn+(Mn+=Ti4+,Ga3+);所述的金属盐溶液为Ti(SO4)2或GaCl3The 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 .

膦基离子液体修饰纳米复合材料的制备方法,包括以下步骤:The preparation method of phosphine-based ionic liquid modified nano composite material comprises the following steps:

(1)将基底材料A分散在无水甲苯中,加入3-氯丙基三乙氧基硅烷,在N2气氛下连续搅拌、加热,得到氯丙基修饰的纳米材料,即A-CP,洗涤、烘干;所述的基底材料A为纳米二氧化硅(nSiO2)、磁性核壳结构纳米二氧化硅(Fe3O4@nSiO2)或介孔二氧化硅包覆的石墨烯(G@mSiO2)中任一种。(2)将PEI溶解在无水乙醇中,搅拌均匀后,向该溶液中加入上述得到的材料A-CP,搅拌、加热后,将PEI接枝到上述所得的A-CP表面,得到材料A@PEI;(3)将得到的材料A@PEI分散在无水甲苯中,然后向其中加入过量溴代烷基磷酸酯,搅拌、加热反应。洗涤、烘干,即可得到膦基功能化离子液体修饰材料,即A@PEI-PFIL;所述的溴代烷基磷酸酯为以下3种任意一种:二乙基(3-溴丙基)磷酸酯、二乙基(4-溴丁基)磷酸酯和二乙基(5-溴戊基)磷酸酯;(4)将得到的材料A@PEI-PFIL分散于去离子水稀释2倍的浓氢溴酸中,搅拌、加热后,用NaOH溶液(PH=11)进行中和,烘干;(5)将步骤(4)得到的材料分散于金属盐溶液中,在37℃下反应2h,便可得到固定金属离子的亲和材料,洗涤、烘干后,即得纳米材料A@PEI-PFIL-Mn+(Mn+=Ti4+,Ga3+);所述的金属盐溶液为Ti(SO4)2或GaCl3(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 .

进一步的,所述步骤(1)中,反应的温度85℃,反应时间为14h。Further, in the step (1), the reaction temperature is 85° C., and the reaction time is 14 hours.

进一步的,所述步骤(2)中,反应的温度80℃,反应时间为24h。Further, in the step (2), the reaction temperature is 80° C., and the reaction time is 24 hours.

进一步的,所述步骤(3)中,反应的温度110℃,反应时间为16h。Further, in the step (3), the reaction temperature is 110° C., and the reaction time is 16 hours.

进一步的,所述步骤(1)、(2)和(3)洗涤液均为乙醇。Further, the washing solutions in steps (1), (2) and (3) are all ethanol.

本发明中,膦基离子液体修饰纳米复合材料在富集磷酸化肽中的应用:将上述膦基离子液体修饰纳米复合材料用于富集磷酸化肽。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.

本发明的原理如下:本发明用溴代烷基磷酸酯季铵化经聚乙烯亚胺改性的纳米材料,将有机磷功能离子液体交联,修饰到基底材料上,得到聚乙烯亚胺改性的纳米材料,即A@PEI-PFIL;在酸化处理后,在有机磷酸基团上修饰金属离子(Mn+),得到A@PEI-PFIL-Mn+固定金属离子亲和色谱材料。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.

本发明通过改变基底材料A和溴代烷基磷酸酯的组合,即:①选择纳米二氧化硅、二氧化硅包覆磁球纳米颗粒和介孔二氧化硅包覆石墨烯与二乙基(3-溴丙基)磷酸酯纳米复合材料,②纳米二氧化硅、二氧化硅包覆磁球纳米颗粒和介孔二氧化硅包覆石墨烯与二乙基(4-溴丁基)磷酸酯纳米复合材料,③纳米二氧化硅、二氧化硅包覆磁球纳米颗粒和介孔二氧化硅包覆石墨烯与二乙基(5-溴戊基)磷酸酯纳米复合材料,制备了九种不同基底与不同配体材料的IMAC吸附剂,即A@PEI-PFIL-Mn+(其中,A=nSiO2、Fe3O4@nSiO2或G@mSiO2PFIL=由二乙基(3-溴丙基)磷酸酯、二乙基(4-溴丁基)磷酸酯或二乙基(5-溴戊基)磷酸酯,Mn+=Ti4+,Ga3+)。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+ ).

与现有技术相比,本发明具有以下有益效果:(1)本发明的修饰流程比较简单,易于操作,且不会改变基底材料的形貌,制备的材料有着良好的环境稳定性和可重复使用性。(2)本发明中所合成的固定金属离子亲和材料:A@PEI-PFIL-Mn+(Mn+=Ti4+,Ga3+)为IMAC型吸附材料,利用磷酸化肽与固定金属离子的亲和性,可用于磷酸化肽的特异性富集,合成的材料有效解决了富集选择性不足、吸附比容量低,蛋白排阻效应差等难题,可用于复杂生物样品中磷酸化肽的富集。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.

附图说明Description of drawings

图1为纳米复合材料A@PEI-PFIL-Mn+(其中,A=nSiO2、Fe3O4@nSiO2或G@mSiO2;PFIL=由二乙基(3-溴丙基)磷酸酯、二乙基(4-溴丁基)磷酸酯或二乙基(5-溴戊基)磷酸酯,Mn+=Ti4+,Ga3+)合成流程图。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.

图2a-图2j为β-酪蛋白酶解液(200fmol/μL)的质谱图;磷酸化肽信号用*表示,去磷酸残基则用#表示,其中: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:

图2a为β-酪蛋白酶解液的直接检测图;Figure 2a is a direct detection figure of β-casein hydrolyzate;

图2b为β-酪蛋白酶解液经过nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(5-溴戊基)磷酸酯)处理后的质谱图;Figure 2b is the mass spectrum of the β-casein hydrolyzate treated with nSiO 2 @PEI-PFIL-Ti 4+ (PFIL=diethyl(5-bromopentyl)phosphate);

图2c为β-酪蛋白酶解液经过nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(4-溴丁基)磷酸酯)处理后的质谱图;Figure 2c is the mass spectrum of β-casein hydrolyzate treated with nSiO 2 @PEI-PFIL-Ti 4+ (PFIL=diethyl(4-bromobutyl)phosphate);

图2d为β-酪蛋白酶解液经过nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(3-溴丙基)磷酸酯)处理后的质谱图;Figure 2d is the mass spectrum of the β-casein hydrolyzate treated with nSiO 2 @PEI-PFIL-Ti 4+ (PFIL=diethyl(3-bromopropyl)phosphate);

图2e为β-酪蛋白酶解液经过Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(5-溴戊基)磷酸酯)处理后的质谱图;Figure 2e is the mass spectrogram of β-casein hydrolyzate treated with Fe3O4@nSiO 2 @PEI-PFIL-Ti 4+ (PFIL=diethyl(5-bromopentyl)phosphate);

图2f为β-酪蛋白酶解液经过Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(4-溴丁基)磷酸酯)处理后的质谱图;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);

图2g为β-酪蛋白酶解液经过Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(3-溴丙基)磷酸酯)处理后的质谱图;Figure 2g is the mass spectrum of β-casein hydrolyzate treated with Fe3O4@nSiO 2 @PEI-PFIL-Ti 4+ (PFIL=diethyl(3-bromopropyl)phosphate);

图2h为β-酪蛋白酶解液经过G@mSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(5-溴戊基)磷酸酯)处理后的质谱图;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);

图2i为β-酪蛋白酶解液经过G@mSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(4-溴丁基)磷酸酯)处理后的质谱图;Figure 2i is the mass spectrum of β-casein hydrolyzate treated with G@mSiO 2 @PEI-PFIL-Ti 4+ (PFIL=diethyl(4-bromobutyl)phosphate);

图2j为β-酪蛋白酶解液经过G@mSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(3-溴丙基)磷酸酯)处理后的质谱图。Fig. 2j is the mass spectrum of β-casein hydrolyzate treated with G@mSiO 2 @PEI-PFIL-Ti 4+ (PFIL=diethyl(3-bromopropyl)phosphate).

图3为β-酪蛋白酶解液用G@mSiO2@PEI-PFIL-Ga3+处理后的质谱图;PFIL=二乙基(3-溴丙基)磷酸酯,磷酸化肽信号用*表示,去磷酸残基则用#表示。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 #.

图4a-图4b为β-酪蛋白和牛血清蛋白BSA的酶解混合液(摩尔比为1:5000)的质谱图;PFIL=二乙基(3-溴丙基)磷酸酯,磷酸化肽信号用*表示,去磷酸残基则用#表示;其中: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:

图4a为G@mSiO2@PEI-PFIL-Ga3+处理后的质谱图;Figure 4a is the mass spectrum after G@mSiO 2 @PEI-PFIL-Ga 3+ treatment;

图4b为G@mSiO2@PEI-PFIL-Ti4+处理后的质谱图。Figure 4b is the mass spectrum after G@mSiO 2 @PEI-PFIL-Ti 4+ treatment.

图5为β-酪蛋(1.43pmol)和牛血清蛋白BSA的酶解混合液(摩尔比为1:12000)在G@mSiO2@PEI-PFIL-Ti4+处理后的质谱图;PFIL=二乙基(3-溴丙基)磷酸酯,磷酸化肽信号用*表示,去磷酸残基则用#表示。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 #.

图6a-图6d为唾液的质谱图;PFIL=二乙基(3-溴丙基)磷酸酯,磷酸化肽信号用*和加点数字或常规数字表示;其中: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:

图6a为唾液样品直接分析的质谱图;Fig. 6a is the mass spectrogram of direct analysis of saliva sample;

图6b为nSiO2@PEI-PFIL-Ti4+处理后的质谱图;Figure 6b is the mass spectrum after nSiO 2 @PEI-PFIL-Ti 4+ treatment;

图6c为Fe3O4@nSiO2@PEI-PFIL-Ti4+处理后的质谱图;Figure 6c is the mass spectrum after Fe 3 O 4 @nSiO 2 @PEI-PFIL-Ti 4+ treatment;

图6d为G@mSiO2@PEI-PFIL-Ti4+处理后的质谱图。Figure 6d is the mass spectrum after G@mSiO 2 @PEI-PFIL-Ti 4+ treatment.

图7a-图7b为吸附剂G@mSiO2@PEI-PFIL-Ti4+蛋白排阻的质谱图;PFIL=二乙基(3-溴丙基)磷酸盐,磷酸化肽信号用*表示,去磷酸残基则用#表示;其中: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:

图7a为β-casein:β-caseinprotein:BSAprotein=1:1000:1000的质谱图;Figure 7a is the mass spectrum of β-casein:β-caseinprotein:BSAprotein=1:1000:1000;

图7b为β-casein:β-caseinprotein:BSAprotein=1:2000:2000的质谱图。Figure 7b is the mass spectrum of β-casein:β-caseinprotein:BSAprotein=1:2000:2000.

图8a-图8c为β-酪蛋白酶解液的质谱图;磷酸化肽信号用*表示,去磷酸残基则用#表示;其中: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:

图8a为G@mSiO2@PEI-PFIL-Ti4+对β-酪蛋白酶解液第1次富集的质谱图;Figure 8a is the mass spectrum of the first enrichment of β-casein hydrolyzate by G@mSiO 2 @PEI-PFIL-Ti 4+ ;

图8b为G@mSiO2@PEI-PFIL-Ti4+对β-酪蛋白酶解液重复使用5次富集的质谱图;Figure 8b is the mass spectrogram of G@mSiO 2 @PEI-PFIL-Ti 4+ enriched β-casein hydrolyzate for 5 times;

图8c为G@mSiO2@PEI-PFIL-Ti4+对β-酪蛋白酶解液重复使用10次富集的质谱图。Fig. 8c is the mass spectrum of G@mSiO 2 @PEI-PFIL-Ti 4+ enriched β-casein hydrolyzate for 10 times.

具体实施方式Detailed ways

下面结合附图与具体实施方式对本发明作进一步详细描述。The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

以下实施方式中,自始至终相同或类似的标号表示相同或类似的原件或具有相同或类似功能的原件,以下通过参考附图描述的实施方式是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。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.

实施例1:其中的A=nSiO2,PFIL=二乙基(5-溴戊基)磷酸酯。Example 1: where A=nSiO 2 , PFIL=diethyl(5-bromopentyl)phosphate.

实施例2:其中的A=nSiO2,PFIL=二乙基(4-溴丁基)磷酸酯。Example 2: where A=nSiO 2 , PFIL=diethyl(4-bromobutyl)phosphate.

实施例3:其中的A=nSiO2,PFIL=二乙基(3-溴丙基)磷酸酯。Example 3: where A=nSiO 2 , PFIL=diethyl(3-bromopropyl)phosphate.

实施例4:其中的A=Fe3O4@nSiO2,PFIL=二乙基(5-溴戊基)磷酸酯。Example 4: where A=Fe 3 O 4 @nSiO 2 , PFIL=diethyl(5-bromopentyl)phosphate.

实施例5:其中的A=Fe3O4@nSiO2,PFIL=二乙基(4-溴丁基)磷酸酯。Example 5: where A=Fe 3 O 4 @nSiO 2 , PFIL=diethyl(4-bromobutyl)phosphate.

实施例6:其中的A=Fe3O4@nSiO2,PFIL=二乙基(3-溴丙基)磷酸酯。Example 6: where A=Fe 3 O 4 @nSiO 2 , PFIL=diethyl(3-bromopropyl)phosphate.

实施例7:其中的A=G@mSiO2,PFIL=二乙基(5-溴戊基)磷酸酯。Example 7: where A=G@mSiO 2 , PFIL=diethyl(5-bromopentyl)phosphate.

实施例8:其中的A=G@mSiO2,PFIL=二乙基(4-溴丁基)磷酸酯。Example 8: where A=G@mSiO 2 , PFIL=diethyl(4-bromobutyl)phosphate.

实施例9:其中的A=G@mSiO2,PFIL=二乙基(3-溴丙基)磷酸酯。Example 9: where A=G@mSiO 2 , PFIL=diethyl(3-bromopropyl)phosphate.

以上9种实施方式中,区别在于基底材料A和配体PFIL的不同,制备方法相同:首先制备氯丙基修饰的纳米复合物,得到A-CP,在上述制备的纳米复合物表面上接上聚乙烯亚胺,再用溴代烷基磷酸酯与聚乙烯亚胺进行交联季铵化,修饰到其表面,即得到A@PEI-PFIL;通过酸化处理后,把金属离子固定在有机磷酸基团上,得到了固定金属离子亲和吸附剂A@PEI-PFIL-Mn+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.

具体的制备方法如下:Concrete preparation method is as follows:

(1)氯丙基修饰的纳米材料的制备,其制备方法如下:将200mg基底材料分散在30mL的无水甲苯中,超声10分钟后,向其中加入0.6mL 3-氯丙基三乙氧基硅烷,在氮气氛围保护下,加热85℃,搅拌反应14h。反应结束后,离心分离固体,乙醇洗涤几次,烘干,即得到A-CP。(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)A@PEI的制备:将1g聚乙烯亚胺溶解在25mL的无水乙醇中,搅拌10分钟后,把200mg的A-CP分散在聚乙烯亚胺的无水乙醇溶液中,进行超声10分钟,加热至80℃,搅拌反应24h。反应结束后,离心分离固体,依次用去离子水和乙醇洗涤几次,烘干,即得到A@PEI。(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)A@PEI-PFIL的制备:将0.5g的溴代烷基磷酸酯滴入15mL的无水甲苯中,超声十分钟,将100mg的A@PEI分散在含溴代烷基磷酸酯的无水甲苯溶液中,进行10分钟的超声,在110℃的油浴中磁力搅拌16h。反应结束后,离心分离固体,乙醇洗涤几次,烘干,即得到A@PEI-PFIL。(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)A@PEI-PFIL的酸化处理:将得到的A@PEI-PFIL分散在6mL稀释2倍后的浓氢溴酸中,在110℃油浴中磁力搅拌2h。反应结束后,用氢氧化钠溶液(pH=11)中和后,再用去离子水洗涤至中性,烘干,即可得到钠盐A@PEI-PFIL-Na+(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)A@PEI-PFIL-Mn+的制备:将经过酸处理后的A@PEI-PFIL-Na+分散于15mL0.1 M金属盐溶液中,室温下震荡2h。反应结束后,离心分离固体后,依次用去离子水和乙醇洗涤,85℃下烘干,即可得到A@PEI-PFIL-Mn+。所述金属盐溶液为Ti(SO4)2或GaCl3溶液。(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.

实验测试及对附图的说明如下:Experimental tests and descriptions of the accompanying drawings are as follows:

(1)为了考察3种不同基底和3种不同配体的纳米材料在修饰钛离子(A@PEI-PFIL-Ti4+)后对磷酸化肽富集的效果,从而确定不同基底材料对富集的影响,我们比较了九种A@PEI-PFIL-Ti4+吸附剂对标准蛋白β-酪蛋白酶解液中磷酸化肽的富集效果。(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.

将5mgβ-酪蛋白溶解于1mL 25mM碳酸氢氨缓冲溶液(pH=8)中;向混合溶液中加入胰蛋白酶(胰蛋白酶与底物的质量比为1:50),在37℃条件下反应12h。酶解后的产物存放于-20℃的冰箱中待用。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.

为了比较九种不同基底的IMAC吸附剂A@PEI-PFIL-Ti4+对磷酸化肽的富集效果,我们首先选择标准蛋白β-酪蛋白作为富集的样品。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.

分别称取2mg 3种不同基底和3种不同配体的吸附剂于0.8mL的富集缓冲液(50%ACN,0.1%TFA,v/v)中,超声10分钟后,取出100μL的分散液用于富集实验,向100μL分散液中加入1μL的标准肽酶解液(200fmol/μL)。然后,将混合液置于恒温金属浴中,在37℃条件下震荡30min,离心或磁性分离固体再用富集缓冲液洗涤固体材料三次。最后,用10μL 0.4M的氨水分散洗涤后的固体材料,在37℃条件下震荡15min,进行三分钟的离心,取上清液5μL与5μL基质溶液(饱和DHB溶液,包含50%ACN和0.1%TFA)混合后,取1μL的混合液滴于MALDI的靶板上,在空气中干燥后进行MALDI-TOF MS分析。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.

质谱检测结果如图2系列所示,图2a为样品不经过处理,直接进行质谱分析的结果,从图2a中可以看出几乎没有磷酸化肽的信号;样品经过九种吸附剂处理后,得到的分析结果如图2中b-j所示,样品在经过nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(5-溴戊基)磷酸酯)、nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(4-溴丁基)磷酸酯)、nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(3-溴丙基)磷酸酯)、Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(5-溴戊基)磷酸酯)、Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(4-溴丁基)磷酸酯)、Fe3O4@nSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(3-溴丙基)磷酸酯)、G@mSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(5-溴戊基)磷酸酯)、G@mSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(4-溴丁基)磷酸酯)和G@mSiO2@PEI-PFIL-Ti4+(PFIL=二乙基(3-溴丙基)磷酸酯)处理分别可以检测到4、3、5、2、4、3、5、5和5个磷酸化肽信号峰以及对应的1、0、1、0、1、1、1、2和2个去磷酸残基信号峰。从质谱结果可知,磷酸化肽的信号峰强度较大,且背景较干净,实验结果表明这九种吸附剂均可以有效地富集磷酸化肽;从磷酸化肽信号个数以及杂峰的情况综合分析,以G@mSiO2为基底、配体PFIL=二乙基(3-溴丙基)磷酸酯的吸附剂G@mSiO2@PEI-PFIL-Ti4+表现出更好的特异性,其原因可能是由于介孔二氧化硅的比表面积大,可以修饰上更多亲水性的聚乙烯亚胺,进行修饰上更多的磷氧基团来固定更多的金属离子,则富集到更多的磷酸化肽。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)为了研究不同的金属离子对富集磷酸化肽的效果,我们将不同金属离子(Ga3+、Ti4+)固定于基底G@mSiO2、配体PFIL=二乙基(3-溴丙基)磷酸酯上,比较固定不同金属离子的材料G@mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+)对β-酪蛋白酶解液中磷酸化肽的富集效果。(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 .

分别称取2mg两种吸附剂G@mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+)于0.8mL的富集缓冲液(50%ACN,0.1%TFA,v/v)中,超声分散后,取出100μL的分散液用于富集实验,向分散液中加入1μL的标准肽酶解液(200fmol/μL)。然后,将混合液置于恒温金属浴中,在37℃条件下震荡30min。反应结束后,离心分离固体并用富集缓冲液洗涤固体材料三次。最后,用10μL0.4M的氨水分散洗涤后的固体材料,在37℃条件下震荡15min,离心后取上清液5μL,与5μL基质溶液(饱和DHB溶液,包含50%ACN和0.1%TFA)混合后,取1μL的混合液滴于MALDI的靶板上,在空气中干燥后进行MALDI-TOF MS分析。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.

质谱分析的对比结果如图3和图2j所示。样品分别经过两种吸附剂G@mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+)处理后,可以观察到图3检测到了1个磷酸化肽信号峰,且背景基线相对于材料G@mSiO2@PEI-PFIL-Ti4+(图2j)较高,样品经G@mSiO2@PEI-PFIL-Ti4+(图2j)处理后,可以检测到5个磷酸化肽的信号峰,从谱图背景及检测到磷酸化肽信号峰个数可知,材料G@mSiO2@PEI-PFIL-Ti4+能够更好地富集多磷酸化肽。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)为了进一步研究不同的金属离子对磷酸化肽特异性富集的效果,比较了固定不同金属离子的材料G@mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+)对β-酪蛋白和牛血清蛋白BSA酶解混合液(摩尔比为1:5000)中磷酸化肽的富集效果。(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).

1mg牛血清蛋白溶解在0.1mL 50mM碳酸氢氨变性缓冲液中(包含8M尿素),变性后加入0.2mL 0.1M二硫苏糖醇(DTT)溶液,并在37℃条件下反应30min,还原蛋白质中的二硫键,然后再加入0.2mL 0.2M碘乙酰胺(IAA)溶液,于室温条件下避光反应30min,使还原的巯基烷基化;将上述产物用50mM碳酸氢氨缓冲溶液(pH=8.3)稀释至1mL;向混合溶液中加入胰蛋白酶(胰蛋白酶与底物的质量比为1:50),在37℃条件下反应16h。酶解后的产物存放于-20℃的冰箱中待用。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.

分别称取2mg两种吸附剂G@mSiO2@PEI-PFIL-Mn+(Ga3+、Ti4+)于1mL的富集缓冲液(50%ACN,0.1%TFA,v/v)中,超声分散后,取出100μL的分散液用于富集实验,向分散液中加入100μL的蛋白酶解混合液。然后,将混合液置于恒温金属浴中,在37℃条件下震荡30min,离心分离固体并用富集缓冲液洗涤固体材料三次。反应结束后,用10μL 0.4M的氨水分散洗涤后的固体材料,在37℃条件下震荡15min,离心后取上清液5μL,与5μL基质溶液(饱和DHB溶液,包含50%ACN和0.1%TFA)混合后,取1μL的混合液滴于MALDI的靶板上,在空气中干燥后进行MALDI-TOF MS分析。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.

分析结果如图4系列所示,图4a为G@mSiO2@PEI-PFIL-Ga3+处理样品后,可以检测到5个磷酸化肽信号峰,但存在一些非磷酸化肽信号,且基线较高;图4b为G@mSiO2@PEI-PFIL-Ti4+处理样品后,可以观察到5个磷酸化肽和1个去磷酸残基的信号,存在较少的非磷酸化肽,背景相对干净。所以,从两个富集样品的富集结果综合分析,G@mSiO2@PEI-PFIL-Ti4+对磷酸化肽富集具有更好的特异性。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)为了更好地评估G@mSiO2@PEI-PFIL-Ti4+纳米复合物对磷酸化肽的吸附能力,我们选择更复杂的蛋白酶解液作为吸附样品,即继续提高β-酪蛋白和牛血清蛋白BSA酶解混合液中BSA酶解液的摩尔比(摩尔比为1:12000)。(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).

称取2mg G@mSiO2@PEI-PFIL-Ti4+于0.8mL的富集缓冲液(50%ACN,1%TFA,v/v)中,超声分散后,取出100μL的分散液用于富集实验,向分散液中加入240μL的蛋白酶解混合液(其中,β-酪蛋白含量为1.43pmol)。然后,将混合液置于恒温金属浴中,在37℃条件下震荡30min,离心分离固体并用富集缓冲液洗涤固体材料三次。最后,用10μL 0.4M的氨水分散洗涤后的固体材料,在37℃条件下震荡15min,离心后取上清液5μL,与5μL基质溶液(饱和DHB溶液,包含50%ACN和0.1%TFA)混合后,取1μL的混合液滴于MALDI的靶板上,在空气中干燥后进行MALDI-TOF MS分析。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.

质谱检测结果如图5所示,在经过G@mSiO2@PEI-PFIL-Ti4+富集后,在谱图中可以发现基线稍微升高了一些,但仍然可以观察到6个磷酸化肽信号,且磷酸化肽信号主导整个质谱图,且磷酸化肽的相对强度较高,所以,材料G@mSiO2@PEI-PFIL-Ti4+对磷酸化肽富集具有良好的特异性。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)3个不同基底的富集材料A@PEI-PFIL-Ti4+(A=nSiO2、Fe3O4@nSiO2和G@mSiO2)对唾液中内源性磷酸化肽的富集;PFIL=二乙基(3-溴丙基)磷酸酯。(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.

分别称取2mg三种吸附剂nSiO2@PEI-PFIL-Ti4+、Fe3O4@nSiO2@PEI-PFIL-Ti4+和G@mSiO2@PEI-PFIL-Ti4+于0.8mL的富集缓冲液(50%ACN,0.1%TFA,v/v)中,超声分散后,取出100μL的分散液用于富集实验,向分散液中加入20μL的唾液样品。然后,将混合液置于恒温金属浴中,在37℃条件下震荡30min。反应结束后,离心分离固体并用富集缓冲液洗涤固体材料三次。最后,用10μL 0.4M的氨水分散洗涤后的固体材料,在37℃条件下震荡15min,离心后取上清液5μL,与5μL基质溶液(饱和DHB溶液,包含50%ACN和0.1%TFA)混合后,取1μL的混合液滴于MALDI的靶板上,在空气中干燥后进行MALDI-TOF MS分析。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.

分析结果如图6系列所示,图6a为唾液样品直接经质谱分析后的质谱图,从图中可以观察到,非磷酸化肽和杂质信号峰主导整个图谱;nSiO2@PEI-PFIL-Ti4+(图6b)、Fe3O4@nSiO2@PEI-PFIL-Ti4+(图6c)和G@mSiO2@PEI-PFIL-Ti4+(图6d)3种吸附剂对样品处理后,分别可以检测到的磷酸化肽的信号峰个数为25、16和27,表明这三种不同基底的材料均可以用以唾液中内源性磷酸化肽的富集。从质谱分析结果可知,图6b富集到相对较少的内源性磷酸化肽,且非磷酸化肽的信号较强,图6c和6d都富集到较多数量的磷酸化肽,但图6d富集到的磷酸化肽更为清晰的显示在质谱图中,以上分析结果表明,3种不同基底的吸附剂对磷酸化肽的富集均表现出较好的亲和性,且G@mSiO2@PEI-PFIL-Ti4+对唾液中内源性磷酸化肽富集的特异性最佳。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)为了表明基于材料设计时介孔二氧化硅的尺寸排阻效应,我们利用材料G@mSiO2@PEI-PFIL-Ti4+对β-酪蛋白酶解液(β-casein)、β-酪蛋白(β-casein protein)、牛血清蛋白(BSAprotein)在以一定的质量比进行了富集实验。(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.

称取2mg吸附剂G@mSiO2@PEI-PFIL-Ti4+于0.8mL的富集缓冲液(50%ACN,0.1%TFA,v/v)中,超声分散后,取出100μL的分散液用于富集实验,向分散液中分别加入了10.1μL和19.2μLβ-酪蛋白酶解液(β-casein)、β-酪蛋白(β-caseinprotein)、牛血清蛋白(BSAprotein)混合溶液。然后,将混合液置于恒温金属浴中,在37℃条件下震荡30min。反应结束后,离心分离固体并用富集缓冲液洗涤固体材料三次。最后,用10μL 0.4M的氨水分散洗涤后的固体材料,在37℃条件下震荡15min,离心后取上清液5μL,与5μL基质溶液(饱和DHB溶液,包含50%ACN和0.1%TFA)混合后,取1μL的混合液滴于MALDI的靶板上,在空气中干燥后进行MALDI-TOF MS分析;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;

检测结果如图7系列所示,其中,图7a和7b为材料G@mSiO2@PEI-PFIL-Ti4+分别对β-酪蛋白酶解液(β-casein):β-酪蛋白(β-caseinprotein):牛血清蛋白蛋白(BSAprotein)β-酪蛋白酶解=1:1000:1000,β-酪蛋白酶解液(β-casein):β-酪蛋白(β-caseinprotein):牛血清蛋白(BSAprotein)β-酪蛋白酶解=1:2000:2000检测结果,从图中可以看出,在图7a质量比为1:1000:1000时,可以检测到4个磷酸化肽的信号峰和2个去磷酸残基信号峰,且峰信号有着很大的强度。在图7b质量比为1:2000:2000时,仍然可以检测磷酸化肽信号峰的个数并没有减少,且谱图背景比较干净,磷酸化肽峰占主导地位,从结果分析可得该吸附剂G@mSiO2@PEI-PFIL-Ti4+能够阻挡大量的大分子量的蛋白质,具有很好的蛋白尺寸排阻效应,在内源性磷酸化肽的研究中有着很大的潜力。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)为了表明基于材料设计时的稳定性特性及重复使用性,我们利用材料G@mSiO2@PEI-PFIL-Ti4+对β-酪蛋白酶解液进行了10次重复性富集。(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.

称取2mg吸附剂G@mSiO2@PEI-PFIL-Ti4+于800μL的富集缓冲液(50%ACN,0.1%TFA,v/v)中,超声分散后,取出100μL的分散液用于富集实验,向分散液中加入β-酪蛋白酶解液(200fmol)。然后,将混合液置于恒温金属浴中,在37℃条件下震荡30min,离心分离固体并用富集缓冲液洗涤固体材料三次。最后,用10μL 0.4M的氨水分散洗涤后的固体材料,在37℃条件下震荡15min,离心后取上清液5μL,与5μL基质溶液(饱和DHB溶液,包含50%ACN和0.1%TFA)混合后,取1μL的混合液滴于MALDI的靶板上,在空气中干燥后进行MALDI-TOFMS分析;脱附后的材料用富集缓冲液洗涤三次,然后在相同的条件下进行下一次的富集—脱附循环实验。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.

检测结果如图8系列所示,其中,图8a和8b为材料G@mSiO2@PEI-PFIL-Ti4+分别对β-酪蛋白酶解液第1次和第5次重复使用后富集的检测结果,从图中可以看出,第1次和第5次的富集效果相似;与第1次和第5次检测结果相比,经过重复使用10次富集后(图8c),在质谱图中,可以观察到4个磷酸化肽峰信号,且检测到磷酸化肽的数量没有太大差异。以上结果表明,该材料在多次富集之后,仍然维持着较好的吸附能力,表现着良好的稳定性及重复使用性。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.

本发明的保护范围包括但不限于以上实施方式,本发明的保护范围以权利要求书为准,任何对本技术做出的本领域的技术人员容易想到的替换、变形、改进均落入本发明的保护范围。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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