Efficient enrichment of phosphopeptides by magnetic TiO₂-coated carbon-encapsulated iron nanoparticles.

Titanium dioxide (TiO₂) has been widely used for phosphopeptide enrichment. Several approaches have been reported to produce magnetic TiO₂ affinity probes. In this report, we present a facile approach to immobilize TiO₂ onto poly(acrylic acid)-functionalized magnetic carbon-encapsulated iron nanoparticles as affinity probes for efficient enrichment of phosphopeptides. By using the new magnetic TiO₂ affinity probes, denoted as TiO₂-coated Fe@CNPs, rapid and effective MALDI-TOF MS profiling of phosphopeptides was demonstrated in different model systems such as tryptic digests of β-casein, and complex β-casein/BSA mixture. The TiO₂-coated Fe@CNPs out-performed the commercial TiO₂-coated magnetic beads for detection of phosphopeptides from tryptic digests of β-casein/BSA mixture with a molar ratio of 1:100. The new TiO₂-coated magnetic probes were also proven to be applicable for real life samples. The magnetic TiO₂-coated Fe@CNPs were employed to selectively isolate phosphopeptides from tryptic digests of HeLa cell lysates and out-performed the commercial magnetic TiO₂ beads in the number of identified phosphopeptides and phosphorylation sites. In a 200-μg equivalent of HeLa cell lysates, we identified 1415 unique phosphopeptides and 1093 phosphorylation sites, indicating the good performance of the new approach.