Magnetic binary metal oxides affinity probe for highly selective enrichment of phosphopeptides

In this work, for the first time, binary metal oxides ((Ti-Sn)O4) were integrated into one entity on an atomic scale on magnetic graphene as affinity probe for highly selective enrichment of phosphopeptides. The newly prepared Fe3O4/graphene/(Ti-Sn)O4 (magG/(Ti-Sn)O4) composites gathered the advantages of large specific surface area of graphene, superparamagnetism, and biocompatibility of iron oxide, and enhanced affinity properties of binary metal oxides. The phosphopeptide enrichment efficiency of the magG/(Ti-Sn)O4 composite was investigated, and the results indicated an ultralow detection limit (1 pg/μL or 4.0 × 10(-11) M) and an ultrahigh selectivity (weight ratio of β-casein and BSA reached up to 1:1500). Compared with magnetic affinity probes with single metal oxide (magG/TiO2, magG/SnO2) or the simple physical mixture of magG/TiO2 and magG/SnO2, the magG/(Ti-Sn)O4 composite possessed stronger specificity, higher selectivity and better efficiency; and more importantly, it possessed the ability to enrich both the mono- and multi- phosophorylated peptides, demonstrating the notable features of the novel binary metal oxides affinity probe in the specific and selective enrichment of phosphopeptides. Additionally, by utilizing the magG/(Ti-Sn)O4 composites, a total number of 349 phosphorylation sites on 170 phosphopeptides including 66 monophosphopeptides and 104 multiphosphopeptides were captured and identified from mouse brain, indicating the great potential for their application in phosphoproteomics analysis in the future.