The use of titanium dioxide for selective enrichment of phosphorylated peptides

Tine E. Thingholm, Martin R. Larsen

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review


Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. The phosphopeptides bound to the TiO2 are subsequently eluted from the chromatographic material using an alkaline buffer. TiO2 chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting and concentration of the sample by reversed phase micro-columns prior to downstream MS and LC-MS/MS analysis.

Original languageEnglish
Title of host publicationPhospho-Proteomics : Methods and Protocols
EditorsLouise von Stechow
PublisherHumana Press
Publication date2016
ISBN (Print)978-1-4939-3048-7
ISBN (Electronic)978-1-4939-3049-4
Publication statusPublished - 2016
SeriesMethods in Molecular Biology


  • Mass spectrometry
  • Phosphopeptide enrichment
  • Protein phosphorylation
  • Titanium dioxide chromatography
  • Phosphorylation
  • Humans
  • Computational Biology
  • Workflow
  • Chromatography, High Pressure Liquid
  • Titanium/chemistry
  • Chromatography, Reverse-Phase
  • Tandem Mass Spectrometry
  • Animals
  • Chromatography, Affinity
  • Adsorption
  • Databases, Protein
  • Surface Properties
  • Protein Binding
  • Proteomics/methods
  • Protein Processing, Post-Translational
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • HeLa Cells
  • Phosphopeptides/agonists


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