Mesoporous TiO2 aerogel for selective enrichment of phosphopeptides in rat liver mitochondria

The enrichment of low abundance phosphopeptides before MS analysis is a critical step for in-depth phosphoproteome research. In this study, mesoporous titanium dioxide (TiO(2)) aerogel was prepared by precipitation and supercritical drying. The specific surface area up to 490.7 m(2) g(-1) is achieved by TiO(2) aerogel, much higher than those obtained by commercial TiO(2) nanoparticles and by the latest reported mesoporous TiO(2) spheres. Due to the large specific surface area and the mesoporous structure of the aerogel, the binding capacity for phosphopeptides is six times higher than that of conventional TiO(2) microparticles (173 vs 28 μmol g(-1)). Because of the good compatibility of enrichment procedure with MALDI-TOF-MS and the large binding capacity of TiO(2) aerogel, a detection limit as low as 30 amol for analyzing phosphopeptides in β-casein digest was achieved. TiO(2) aerogel was further applied to enrich phosphopeptides from rat liver mitochondria, and 266 unique phosphopeptides with 340 phosphorylation sites, corresponding to 216 phosphoprotein groups, were identified by triplicate nanoRPLC-ESI-MS/MS runs, with false-positive rate less than 1% at the peptide level. These results demonstrate that TiO(2) aerogel is a kind of promising material for sample pretreatment in the large-scale phosphoproteome study.