Phosphonate-Functionalized Ionic Liquid: A New Surface Modifier Contributing to the Enhanced Enrichment of Phosphorylated Peptides

Phosphopeptide enrichment with high selectivity and detection sensitivity is essential for phosphoproteomic studies and remains a long-standing challenge. In this study, new immobilized metal affinity chromatography nanocomposite adsorbents with a phosphonate-functionalized ionic liquid (PFIL) as a surface modifier are successfully prepared via a reaction sequence of amination, quaternization, phosphonate hydrolyzation, and metal immobilization. Taking advantages of integrated features of a flexible and strong tripodal phosphonate chelator, a hydrophilic ionic liquid linker, a large surface area, and the size-exclusion effect, the resulting nanocomposite G@mSiO2-PFIL-Ti4+ exhibits excellent detection sensitivity to enrich phosphorylated peptides from a tryptic β-casein digest (0.15 fmol), and superior enrichment selectivity to capture phosphorylated peptides from a digest mixture of β-casein and bovine serum albumin (a molar ratio of 1:10,000). Strong immobilization of tripodal chelation to metal ions endows the nanocomposite adsorbent with high tolerance to experimental conditions, and thus excellent reusability of the adsorbent has been achieved without remarkable loss of enrichment efficiency for 10 cycles. Due to the excellent size-exclusion effect, high enrichment specificity of G@mSiO2-PFIL-Ti4+ to phosphopeptides has been observed and 23 endogenous phosphopeptides have been captured from human saliva. In addition, 924 phosphopeptides (enrichment specificity, 56.1%) have been identified from the tryptic digest of mouse brain lysate. Particularly, six of 975 phosphorylation sites were Alzheimer's disease-related hyperphosphorylation sites within tau protein. These results demonstrate that G@mSiO2-PFIL-Ti4+ nanocomposite affinity materials show great application potential for a proteomic study of complicated biological samples.