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

Abstract

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.