A poly(ethylene glycol)-brush decorated magnetic polymer for highly specific enrichment of phosphopeptides

Immobilized metal affinity chromatography (IMAC) is a powerful method in phosphopeptide enrichment. However, the achievement of highly specific enrichment and sensitive detection of phosphopeptide by IMAC is still a big challenge because of the lack of high specificity and large binding capacity of conventional IMAC materials. Here, we report a novel IMAC nanoparticle to dramatically improve the enrichment specificity for the phosphopeptide by introducing a titanium phosphate moiety on a poly(ethylene glycol) methacrylate (PEG) brush decorated Fe3O4@SiO2 core-shell nanoparticle (denoted as Fe3O4@SiO2@PEG-Ti4+ IMAC nanoparticle). The thicker grafting layer of the PEG brushes has a higher chelating capacity of titanium ions. Due to the combination of the superior nonfouling property and the enhanced binding capacity of the grafted PEG brushes, the Fe3O4@SiO2@PEG-Ti4+ IMAC nanoparticle demonstrated a high phosphopeptide recovery (over 70%) and low limit of detection (0.5 fmol), along with an exceptional great specificity to capture phosphopeptides from a tryptic digest of the mixture of a nonphosphorylated protein BSA and a phosphorylated protein alpha-casein with molar ratios of BSA/alpha-casein up to 2000 : 1. In the analysis of a real complex biological sample, the tryptic digests of Arabidopsis, 2447 unique phosphopeptides have been identified, showing a superior performance of the Fe3O4@SiO2@PEG-Ti4+ IMAC nanoparticle than that of Fe3O4@SiO2-Ti4+ (1186) and commercial TiO2 microspheres (961). We believe that the PEG decoration for IMAC materials will be a convenient approach to significantly improve the specificity and the binding capacity of phosphopeptide enrichment.