Fe3O4/TiO2 Core/Shell Nanoparticles as Affinity Probes for the Analysis of Phosphopeptides Using TiO2 Surface-Assisted Laser Desorption/Ionization Mass Spectrometry

Abstract

Columns packed with microsized titanium dioxide particles have been used effectively as precolumns for enriching phosphopeptides from complex mixtures. Nanosized titanium dioxide particles have a higher specific surface area and, hence, potentially higher trapping capacities toward phosphopeptides than do microsized particles. Thus, in this study, we employed TiO₂-coated magnetic (Fe₃O₄/TiO₂ core/shell) nanoparticles to selectively concentrate phosphopeptides from protein digest products. Because of their magnetic properties, the Fe₃O₄/TiO₂ core/shell nanoparticles that are conjugated to the target peptides can be isolated readily from the sample solutions by employing a magnetic field. In this paper, we also demonstrate that the Fe₃O₄/TiO₂ core/shell nanoparticles behave as an effective SALDI matrix: our upper detectable mass limit was ∼24 000 Da, whereas the detection limit for peptides was in the low-femtomole range. That is to say, the target analytes trapped by the Fe₃O₄/TiO₂ nanoparticles can be identified by introducing the particles directly into the mass spectrometer for TiO₂-SALDI-MS analysis without the need for any further treatment. For example, elution steps are not necessary when using this approach. In addition, the trapping selectivity of these Fe₃O₄/TiO₂ nanoparticles toward phosphopeptides was quite good. These properties combine to result in the low detection limits. The lowest detectable concentration of phosphopeptides that we analyzed using this approach was 500 pM for a 100-μL tryptic digest solution of β-casein; this level is much lower than that which can be obtained using any other currently available method.