Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Manuela Cuccurullo, Gitta Schlosser, Giuseppina Cacace, Livia Malorni, Gabriella Pocsfalvi

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26 Citations (Scopus)


Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS3 analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO2). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO2-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for β-casein and three for αS1-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO2-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component αS2-casein by ESI-MS/MS and none for κ-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.

Original languageEnglish
Pages (from-to)1069-1078
Number of pages10
JournalJournal of Mass Spectrometry
Issue number8
Publication statusPublished - Aug 1 2007


  • Bovine milk caseins
  • Phosphopeptide enrichment
  • ZrO

ASJC Scopus subject areas

  • Spectroscopy

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