固定化金属离子亲和发光二氧化硅纳米粒子的制备及其用于磷酸化蛋白免疫印迹标记

Preparation of luminescent silica nanoparticles with immobilized metal ion affinity for labelling phosphorylated proteins in Western Blot

发展了一种能够识别磷酸化蛋白的固定化金属离子亲和发光二氧化硅纳米粒子用于免疫印迹(Western Blot)磷酸化蛋白的标记。首先通过反相微乳液Stöber方法合成了掺杂异硫氰酸荧光素硅烷化衍生物的发光二氧化硅(FITC@SiO_(2))球形纳米粒子,粒子平均粒径为60 nm。然后通过共聚反应在FITC@SiO_(2)纳米粒子表面生成一层聚合物用于保护纳米粒子,并引入N,N-(双羧甲基)-L-赖氨酸功能基团用于螯合金属离子,从而实现固定化金属离子亲和作用。以α-酪蛋白作为实验模型,用高效液相色谱-质谱研究了螯合不同金属离子的发光纳米粒子对磷酸化蛋白的识别作用。结果表明,螯合了Ti^(4+)金属离子的发光二氧化硅FITC@SiO_(2)纳米粒子对α-酪蛋白酶解液中的磷酸化肽段的富集作用最强。利用这种发光二氧化硅FITC@SiO_(2)纳米粒子对磷酸化肽段的特异性识别性能,可用于Western Blot实验中标记磷酸化蛋白的条带。结果显示,α-酪蛋白的电泳条带可以被亲和发光二氧化硅FITC@SiO_(2)纳米粒子标记,而作为对照的牛血清白蛋白则没有被标记。

Protein phosphorylation is an important type of post-translational protein modification.In Western Blot experiment,the assay of phosphoproteins need special phospho antibodies,which are expensive,difficult to preserve,poorly reproducible.To this end,the immobilized metal ion affinity luminescent silica nanoparticles for instead of phospho antibodies were prepared.A layer of polymer was created on the surface of the silica nanoparticles via co-polymerization to protect the nanoparticles and to functionalize them with the immobilized metal ion affinity property to specifically label the phosphorylated proteins in Western Blot assays.The affinity luminescent silica nanoparticles were prepared with the following procedure.First,the sol-gel precursor fluorescein isothiocyanate-3-aminopropyltriethoxysilane(FITC-APTES)with the fluorescent moiety was prepared by modifying APTES with FITC.The luminescent silica nanoparticles(FITC@SiO_(2))were synthesized using the Stöber synthesis method in a reversed microemulsion.Briefly,123.2 mL of cyclohexane,25.6 mL of n-hexanol,and 5.44 mL of deionized water were ultrasonically mixed,and then 28.3 g of Triton X-100 were added and the mixture was magnetically stirred for 15 min to form a clear and transparent microemulsion system.Within 10 min,0.8 mL of FITC-APTES precursor,1.6 mL of tetraethoxysilane(TEOS),and 0.96 mL of concentrated ammonia(25%-27%,mass fraction)were added to the microemulsion,and the mixture was stirred at 24℃for 24 h.After the reaction,the microemulsion system was destroyed by adding 200 mL of ethanol.The resulting FITC@SiO_(2)luminescent silica nanoparticles were centrifuged,and washed three times with ethanol.After dryness,the FITC@SiO_(2)nanoparticles were modified with methacryloxy-propyltrimethoxysilane(MPS)to introduce the double bonds for further modification.The functional monomer nitrilotriacetic acid(NTA)and glycidyl methacrylate(GMA)were copolymerized on the surface of the nanoparticles to convert FITC@SiO_(2)-MPS to FITC@SiO_(2)-MPS-GMA-NTA.The polymer coating of the silica nanoparticles was not only able to protect the silica from hydrolysis,but also to introduce the functional groups of nitrilotriacetic acid,which can chelate with metal ions.Elemental analysis demonstrated that the NTA groups had been bonded to the surface of the nanoparticles via copolymerization.The polymerization did not affect the morphology and fluorescence properties of the nanoparticles.The FITC@SiO_(2)-MPS-GMA-NTA nanoparticles were activated with three different metal ions Zr^(4+),Fe^(3+),and Ti^(4+),for the enrichment of phosphorylated peptides derived formα-casein tryptic digestion.HPLC-MS analysis indicated that the FITC@SiO_(2)-MPS-GMA-NTA-Ti^(4+)nanoparticles are the best for the enrichment of phosphorylated peptides.The FITC@SiO_(2)-MPS-GMA-NTA-Ti^(4+)nanoparticles were used for labelling the phosphorylated proteins in Western Blot experiment.The electrophoretic band ofα-casein could be clearly labeled with the FITC@SiO_(2)-MPS-GMA-NTA-Ti^(4+)nanoparticles,while the bovine albumin band could not be labelled.This indicates that the luminescent FITC@SiO_(2)-MPS-GMA-NTA-Ti^(4+)nanoparticles can be used to label the phosphorylated proteins in Western Blot experiments.