Highly efficient enrichment of phosphopeptides from HeLa cells using hollow magnetic macro/mesoporous TiO₂...

In this work, hollow magnetic macro/mesoporous TiO₂ nanoparticles (denoted as Fe₃O₄@H-fTiO₂) were synthesized by a facile "hydrothermal etching assisted crystallization" route to improve the phosphopeptide enrichment efficiency. The porous nanostructure of TiO₂ shell and large hollow space endowed the Fe₃O₄@H-fTiO₂ with a high surface area (144.71 m² g^-1) and a large pore volume (0.52 cm³ g^-1), which could provide more affinity sites for phosphopeptide enrichment. Besides, the large pore size of TiO₂ 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 TiO₂ with small and deep pores (denoted as Fe₃O₄@H-mTiO₂) and solid magnetic macro/mesoporous TiO₂, the Fe₃O₄@H-fTiO₂ 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 Fe₃O₄@H-fTiO₂ nanoparticles, further demonstrating that the Fe₃O₄@H-fTiO₂ nanoparticles had a high-efficiency performance for phosphopeptide enrichment. Taken together, the Fe₃O₄@H-fTiO₂ nanoparticles will have unique advantages in phosphoproteomics analysis.