Mass spectrometric analysis of mono- and multi-phosphopeptides by selective binding with NiZnFe2O4 magnetic nanoparticles

Selective isolation of mono-and multi-phosphorylated peptides is important for understanding how a graded protein kinase or phosphatase signal can precisely modulate the on and off states of signal transduction pathways. Here we report that metal ions at exposed octahedral sites of nano-ferrites, including Fe3O4, NiFe2O4, ZnFe2O4 and NiZnFe2O4, have distinctly selective coordination abilities with mono-and multi-phosphopeptides. Due to their intrinsic magnetic properties and high surface area to volume ratios, these nanoparticles enable the rapid isolation of mono-and multi-phosphopeptides by an external magnetic field. Model phosphoprotein alpha-casein and two synthesized mono- and di-phosphopeptides have been chosen for proof-of-principle demonstrations, and these nanoparticles have also been applied to phosphoproteome profiling of zebrafish eggs. It is shown that NiZnFe2O4 is highly selective for multi-phosphopeptides. In contrast, Fe3O4, NiFe2O4 and ZnFe2O4 can bind with both mono-and multi-phosphopeptides with relatively stronger affinity towards monophosphopeptides.