A systematic evaluation of nanocast metal oxide microspheres for phosphoproteomics applications

We used the nanocasting technique to prepare a range of different metal oxide microspheres that allow an objective comparison of the different materials. Using samples of different complexity, ranging from synthetic peptides to whole cell lysates, we characterized the binding behavior of these materials. We observed that the phosphoproteome coverage achievable using TiO2, ZrO2, Fe2O3 and In2O3 is comparable in depth, although some differences between individual oxides exist. Single-shot LC-MS/MS experiments on a linear ion trap-orbitrap instrument identified more than 3000 phosphopeptides from the equivalent of 20 μg of HEK cell lysate protein. However, the application of common enrichment protocols to biological samples may still lead to some bias due to an underrepresentation of highly phosphorylated peptides. These peptides are predominantly observed when materials with lower phosphopeptide affinity such as NiO and Co3O4 are used for enrichment and their biological relevance may be underestimated.