Department of Chemistry, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy.
Department of Science and Technological Innovation, Università degli Studi del Piemonte Orientale, Viale Teresa Michel 11, 15121 Alessandria, Italy.
Talanta. 2018 Feb 1;178:274-281. doi: 10.1016/j.talanta.2017.09.010. Epub 2017 Sep 9.
The work describes the preparation of a new magnetic phase for batch enrichment of phosphopeptides. The material exploits the advantages of magnetic solid phase extraction and couples them with the most employed approach for phosphopeptide enrichment, i.e. Ti-IMAC. In order to immobilize Ti ions on the surface of the magnetite nanoparticles, they were first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate was subsequently polymerized from these groups using the "grafting from" approach by the activator regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP) technique. Finally, the glycidyl groups were reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. The prepared material was extensively characterized and subsequently tested for enrichment of a bovine serum albumin mixture with casein to ascertain its potential. With positive results, the new magnetic polymeric material was further employed to set up an enrichment method on yeast protein digest based on shotgun proteomics. The sample to phase ratio was optimized and the best condition compared to a commercial TiO spin column. At the end of the comparison, the new material proved better and could enrich a larger total number of phosphopeptides with increased selectivity. All these conclusions and the test performed on a real complex sample within the final shotgun application further support the applicability of the new material in phosphopeptide analysis of real matrices.
该工作描述了一种新的磁性相的制备，用于批量富集磷酸肽。该材料利用了磁性固相萃取的优势，并将其与最常用的磷酸肽富集方法（Ti-IMAC）相结合。为了将 Ti 离子固定在磁铁矿纳米粒子的表面，首先用二氧化硅壳覆盖它们，然后修饰以暴露表面含溴基团。然后使用电子转移-原子转移自由基聚合（ARGET-ATRP）技术的引发剂再生剂，从这些基团聚合甲基丙烯酸缩水甘油酯。最后，缩水甘油基与亚氨基二乙酸反应，使材料官能化，得到适合配位的部分。所制备的材料进行了广泛的表征，并随后用于牛血清白蛋白与酪蛋白混合物的富集，以确定其潜力。结果为阳性，新的磁性聚合物材料进一步用于基于鸟枪法蛋白质组学的酵母蛋白消化物的富集方法。优化了样品与相的比例，并与商业 TiO 旋转柱进行了比较。在比较结束时，新材料证明更好，可以富集更多具有更高选择性的磷酸肽。所有这些结论以及最终在鸟枪法应用中对实际复杂样品的测试进一步支持了新材料在实际基质中磷酸肽分析中的适用性。
