Highly efficient enrichment of phosphopeptides from HeLa cells using hollow magnetic macro/mesoporous TiO nanoparticles.

In this work, hollow magnetic macro/mesoporous TiO nanoparticles (denoted as FeO@H-fTiO) were synthesized by a facile "hydrothermal etching assisted crystallization" route to improve the phosphopeptide enrichment efficiency. The porous nanostructure of TiO shell and large hollow space endowed the FeO@H-fTiO with a high surface area (144.71 m g) and a large pore volume (0.52 cm g), which could provide more affinity sites for phosphopeptide enrichment. Besides, the large pore size of TiO nanosheets and large hollow space could effectively prevent the "shadow effect", thereby facilitating the diffusion and release of phosphopeptides. Compared with the hollow magnetic mesoporous TiO with small and deep pores (denoted as FeO@H-mTiO) and solid magnetic macro/mesoporous TiO, the FeO@H-fTiO nanoparticles showed a better selectivity (molar ratio of α-casein/BSA up to 1:10000) and a higher sensitivity (0.2 fmol/μL α-casein) for phosphopeptide enrichment. Furthermore, 1485 unique phosphopeptides derived from 660 phosphoproteins were identified from HeLa cell extracts after enrichment with FeO@H-fTiO nanoparticles, further demonstrating that the FeO@H-fTiO nanoparticles had a high-efficiency performance for phosphopeptide enrichment. Taken together, the FeO@H-fTiO nanoparticles will have unique advantages in phosphoproteomics analysis.