Large scale characterization of phosphoproteins requires highly specific methods for purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. Enrichment of phosphopeptides from complex peptide mixtures by IMAC is a popular way to perform phosphoproteome analysis. However, conventional IMAC adsorbents with iminodiacetic acid as the chelating group to immobilize Fe³⁺ lack enough specificity for efficient phosphoproteome analysis. Here we report a novel IMAC adsorbent through Zr⁴⁺ chelation to the phosphonate-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads. The high specificity of Zr⁴⁺-IMAC adsorbent was demonstrated by effectively enriching phosphopeptides from the digest mixture of phosphoprotein (α- or β-casein) and bovine serum albumin with molar ratio at 1:100. Zr⁴⁺-IMAC adsorbent was also successfully applied for the analysis of mouse liver phosphoproteome, resulting in the identification of 153 phosphopeptides (163 phosphorylation sites) from 133 proteins in mouse liver lysate. Significantly more phosphopeptides were identified than by the conventional Fe³⁺-IMAC approach, indicating the excellent performance of the Zr⁴⁺-IMAC approach. The high specificity of Zr⁴⁺-IMAC adsorbent was found to mainly result from the strong interaction between chelating Zr⁴⁺ and phosphate group on phosphopeptides. Enrichment of phosphopeptides by Zr⁴⁺-IMAC provides a powerful approach for large scale phosphoproteome analysis.

The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe³⁺ immobilized metal affinity chromatography (Fe³⁺‐IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe³⁺‐IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.