PAMAM–PMAA brush-functionalized magnetic composite nanospheres: a smart nanoprobe with tunable selectivity for effective enrichment of mono-, multi-, or global phosphopeptides

Abstract

Selective capture of mono-, multi- or global phosphopeptides is significant for the in-depth study of protein kinase or phosphatase signal transduction pathways. However, this study is largely restricted because of the lack of versatile affinity materials with high enrichment capacity and tunable selectivity. Here, we prepared a smart nanoprobe for the selective enrichment of mono-, multi- or global phosphopeptides by introducing polyamidoamine dendrimer (PAMAM)-grafted poly(methacrylic acid) (PMAA) brushes to modified magnetic composite nanospheres (denoted as Fe₃O₄@PDA@PMAA@PAMAM). The practicability of the prepared nanospheres for phosphopeptide enrichment was investigated by using model proteins (α-casein and β-casein) and complex biological samples (nonfat milk and human saliva). The extremely abundant amine groups of PAMAM–PMAA brushes and superparamagnetism of the Fe₃O₄ core endowed the composite nanospheres with high detection sensitivity (1 fmol μL⁻¹), excellent selectivity (1 : 500 molar ratios of β-casein/BSA) and high recyclability (five cycles) towards phosphopeptides. Additionally, based on the tunable bonding ability of the nanospheres towards phosphopeptides with different phosphorylation sites, selective enrichment of mono-, multi- or global phosphopeptides was realized by modulating buffer polarity and acidity, making them ideal new nanoprobes for comprehensive phosphoproteome analysis.