A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes

J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Feb 15;878(5-6):515-24. doi: 10.1016/j.jchromb.2009.12.017. Epub 2009 Dec 22.


The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004; Larsen et al., 2005; Kweon and Hakansson, 2006; Cantin et al., 2007; Collins et al., 2007). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO(2)-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO(2) and ZrO(2) columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / analysis
  • Amino Acid Sequence
  • Anaphase-Promoting Complex-Cyclosome
  • Animals
  • Cattle
  • Chromatography, High Pressure Liquid / methods*
  • DNA-Binding Proteins / analysis
  • HeLa Cells
  • Humans
  • Mass Spectrometry
  • Molecular Sequence Data
  • Multiprotein Complexes / analysis
  • Phosphopeptides / isolation & purification*
  • Phosphorylation
  • Proteins / analysis*
  • Serum Albumin, Bovine / analysis
  • Titanium / chemistry*
  • Ubiquitin-Protein Ligase Complexes / analysis
  • Zirconium / chemistry*


  • DNA-Binding Proteins
  • Multiprotein Complexes
  • Phosphopeptides
  • Proteins
  • condensin complexes
  • titanium dioxide
  • Serum Albumin, Bovine
  • Zirconium
  • Titanium
  • Ubiquitin-Protein Ligase Complexes
  • Anaphase-Promoting Complex-Cyclosome
  • Adenosine Triphosphatases
  • zirconium oxide