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

In this work, hollow magnetic macro/mesoporous TiO 2 nanoparticles (denoted as Fe 3 O 4 @H- f TiO 2 ) were synthesized by a facile “hydrothermal etching assisted crystallization” route to improve the phosphopeptide enrichment efficiency. The porous nanostructure of TiO 2 shell and large hollow space endowed the Fe 3 O 4 @H- f TiO 2 with a high surface area (144.71 m 2 g −1 ) and a large pore volume (0.52 cm 3 g −1 ), which could provide more affinity sites for phosphopeptide enrichment. Besides, the large pore size of TiO 2 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 2 with small and deep pores (denoted as Fe 3 O 4 @H-mTiO 2 ) and solid magnetic macro/mesoporous TiO 2 , the Fe 3 O 4 @H- f TiO 2 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 Fe 3 O 4 @H- f TiO 2 nanoparticles, further demonstrating that the Fe 3 O 4 @H- f TiO 2 nanoparticles had a high-efficiency performance for phosphopeptide enrichment. Taken together, the Fe 3 O 4 @H- f TiO 2 nanoparticles will have unique advantages in phosphoproteomics analysis.