Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis

Abstract

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 Fe 3؉ lack enough specificity for efficient phosphoproteome analysis. Here we report a novel IMAC adsorbent through Zr 4؉ chelation to the phosphonate-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) polymer beads. The high specificity of Zr 4؉ -IMAC adsorbent was demonstrated by effectively enriching phosphopeptides from the digest mixture of phosphoprotein (␣-or ␤-casein) and bovine serum albumin with molar ratio at 1:100. Zr 4؉ -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 Fe 3؉ -IMAC approach, indicating the excellent performance of the Zr 4؉ -IMAC approach. The high specificity of Zr 4؉ -IMAC adsorbent was found to mainly result from the strong interaction between chelating Zr 4؉ and phosphate group on phosphopeptides. Enrichment of phosphopeptides by Zr 4؉ -IMAC provides a powerful approach for large scale phosphoproteome analysis.

ArMone: A Software Suite Specially Designed for Processing and Analysis of Phosphoproteome Data

“…The combination of the results from consecutive MS2 and MS3 spectra can improve ArMone: A Software Suite technical notes the sensitivity and reliability of phosphopeptide identification. 29,30 Recently, we presented a MS 2 /MS 3 target-decoy strategy for confident phosphopeptide identification which integrates the search results from both MS 2 and MS 3 spectra. 18 In MS 2 /MS 3 strategy, MS 2 spectra and MS 3 spectra are separately searched against the same target-decoy database, and the search results are combined before filtering.…”

Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

“…Thus, post-modification is more suitable for the introduction of ionizable groups, such as phosphate groups, onto monolithic pore surface. Feng et al [34] synthesized poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-EDMA) monolith material as the support for ZrP-IMAC. To introduce phosphate groups, epoxide groups on monolith surface were first converted to amino groups by incubation in ammonia solution, followed by reaction of amino groups with POCl 3 .…”

Perspectives of Comprehensive Phosphoproteome Analysis Using Shotgun Strategy

“…However, since most of the phosphopeptides undergo NL of H 3 PO 4 in CID, significant NL ions can be exploited as a marker of phosphopeptides. 14,30 To further fragment the phosphopeptide backbone and obtain fragmentrich mass spectra, a data dependent MS 3 strategy has been widely employed. 31,32 In this method, the significant NL ion which has been generated from MS 2 spectrum is isolated and subjected to an additional CID to generate an MS 3 spectrum.…”