关键词: 免疫印迹, 固定化金属离子亲和, 发光二氧化硅纳米粒子, 磷酸化蛋白标记

Abstract:

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@SiO₂) 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@SiO₂ luminescent silica nanoparticles were centrifuged, and washed three times with ethanol. After dryness, the FITC@SiO₂ 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@SiO₂-MPS to FITC@SiO₂-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@SiO₂-MPS-GMA-NTA nanoparticles were activated with three different metal ions Zr⁴⁺, Fe³⁺, and Ti⁴⁺, for the enrichment of phosphorylated peptides derived form α-casein tryptic digestion. HPLC-MS analysis indicated that the FITC@SiO₂-MPS-GMA-NTA-Ti ⁴⁺ nanoparticles are the best for the enrichment of phosphorylated peptides. The FITC@SiO₂-MPS-GMA-NTA-Ti⁴⁺ nanoparticles were used for labelling the phosphorylated proteins in Western Blot experiment. The electrophoretic band of α-casein could be clearly labeled with the FITC@SiO₂-MPS-GMA-NTA-Ti ⁴⁺ nanoparticles, while the bovine albumin band could not be labelled. This indicates that the luminescent FITC@SiO₂-MPS-GMA-NTA-Ti⁴⁺nanoparticles can be used to label the phosphorylated proteins in Western Blot experiments.

Key words: Western Blot, immobilized metal ion affinity, luminescent silica nanoparticles, phosphorylated protein labelling