EJMS protocol: systematic studies on TiO2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometry-based phosphopeptide stability estimations?

Eur J Mass Spectrom (Chichester). 2011;17(5):507-23. doi: 10.1255/ejms.1134.


There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetophenones / chemistry
  • Amino Acid Sequence
  • Caseins / chemistry
  • Citric Acid
  • Crystallization
  • Electrophoresis, Gel, Two-Dimensional
  • Electrophoresis, Polyacrylamide Gel
  • Gels
  • Gentisates / chemistry
  • Molecular Sequence Data
  • Phosphopeptides / analysis
  • Phosphopeptides / isolation & purification*
  • Phosphorylation
  • Repressor Proteins / chemistry
  • Solutions
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Stathmin / chemistry
  • Titanium* / chemistry
  • Tripartite Motif-Containing Protein 28


  • Acetophenones
  • Caseins
  • Gels
  • Gentisates
  • Phosphopeptides
  • Repressor Proteins
  • Solutions
  • Stathmin
  • Stmn1 protein, rat
  • titanium dioxide
  • Citric Acid
  • 2,4,6-trihydroxyacetophenone
  • Titanium
  • TRIM28 protein, human
  • Tripartite Motif-Containing Protein 28
  • 2,5-dihydroxybenzoic acid