Novel core–shell Cerium(IV)-immobilized magnetic polymeric microspheres for selective enrichment and rapid separation of phosphopeptides

In this work, novel magnetic polymeric core–shell structured microspheres with immobilized Ce(IV), Fe 3 O 4 @SiO 2 @PVPA-Ce(IV), were designed rationally and synthesized successfully via a facile route for the first time. Magnetic Fe 3 O 4 @SiO 2 microspheres were first prepared by directly coating a thin layer of silica onto Fe 3 O 4 magnetic particles using a sol–gel method, a poly(vinylphosphonic acid) (PVPA) shell was then coated on the Fe 3 O 4 @SiO 2 microspheres to form Fe 3 O 4 @SiO 2 @PVPA microspheres through a radical polymerization reaction, and finally Ce(IV) ions were robustly immobilized onto the Fe 3 O 4 @SiO 2 @PVPA microspheres through strong chelation between Ce(IV) ions and phosphate moieties in the PVPA. The applicability of the Fe 3 O 4 @SiO 2 @PVPA-Ce(IV) microspheres for selective enrichment and rapid separation of phosphopeptides from proteolytic digests of standard and real protein samples was investigated. The results demonstrated that the core–shell structured Fe 3 O 4 @SiO 2 @PVPA-Ce(IV) microspheres with abundant Ce(IV) affinity sites and excellent magnetic responsiveness can effectively purify phosphopeptides from complex biosamples for MS detection taking advantage of the rapid magnetic separation and the selective affinity between Ce(IV) ions and phosphate moieties of the phosphopeptides. Furthermore, they can be effectively recycled and show good reusability, and have better performance than commercial TiO 2 beads and homemade Fe 3 O 4 @PMAA-Ce(IV) microspheres. Thus the Fe 3 O 4 @SiO 2 @PVPA-Ce(IV) microspheres can benefit greatly the mass spectrometric qualitative analysis of phosphopeptides in phosphoproteome research.