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A novel magnetic nanomaterial for use in metal ion based affinity chromatography is described. It is based on the chelation between the phosphate groups of phytic acid (PA) and Ti(IV) ions. Due to the large number (6) of phosphate groups of PA, it has a large capacity for Ti(IV) ions. PA was first immobilized on magnetite nanoparticles (PA-MNPs) and then loaded with Ti(IV) ions to obtain the sorbent (Ti-PA-MNPs). The fraction of Ti(IV) ions on the surface of PA-MNPs that is exposed to the solution binds the phosphate groups of phosphopeptides. The bound phosphopeptides can then be magnetically separated. The method was applied to the enrichment of the phosphopeptides in a β-casein tryptic digest. A tryptic digest of bovine serum albumin (BSA) was added at a molar ratio (β-casein to BSA) of 1:2000 to study selectivity. The phosphopeptides were quantified by mass spectrometry. The limit of detection can be as low as 8 × 10

The authors describe a fluorometric method for the determination of the activity and inhibition of protein kinase A (PKA). In the presence of ATP, PKA catalyzes the transfer of phosphate groups from ATP to a peptide, and the generated phosphorylated peptide quenches the fluorescence (measured at excitation/emission peaks of 340/440 nm) of the hydroxyapatite nanoparticles (HAP-NPs). A linear logarithmic relationship of PKA concentrations with fluorescence intensity in the range from 1 to 50 U·L−1 was obtained, and the lower limit of detection (LOD) is 0.5 U·L−1. This is much lower than LODs reported in the literature. The PKA inhibitor H-89 was studied, and the inhibition plot has a sigmoidal shape with a half-maximal inhibitory concentration of around 750 nM of H-89. At a 4.5 nM level of H-89, fluorescence of HAP-NPs fell to levels of no PKA controls, demonstrating that the assay is a viable tool to screen for kinase inhibitors. An assay with Hela cell lysates in combination with forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor used to activate the cellular activity of PKA) resulted in decreased fluorescence of HAP-NPs. This suggests that the assay can be applied for testing in vitro cell kinase activity. In our perception, this method will enable high-throughput screening for kinase-related drugs and fluorometric enzymatic detection in various areas.