The use of titanium dioxide micro-columns to selectively isolate phosphopeptides from proteolytic digests

Tine E Thingholm, Martin R Larsen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Titanium dioxide has very high affinity for phosphopeptides and it has become an efficient alternative to already existing methods for phosphopeptide enrichment from complex samples. Peptide loading in a highly acidic environment in the presence of 2,5-dihydroxybenzoic acid (DHB), phthalic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding from nonphosphorylated peptides. The enriched phosphopeptides bound to the titanium dioxide are subsequently eluted from the micro-column using an alkaline buffer. Titanium dioxide chromatography is extremely tolerant towards most buffers used in biological experiments. It is highly robust and as such it has become one of the methods of choice in large-scale phospho-proteomics. Here we describe the protocol for phosphopeptide enrichment using titanium dioxide chromatography followed by desalting and concentration of the sample by reversed phase chromatography prior to MS analysis.
Original languageEnglish
JournalMethods in Molecular Biology
Volume527
Pages (from-to)57-66
ISSN1064-3745
DOIs
Publication statusPublished - 1. Jan 2009

Keywords

  • Animals
  • Chromatography, Gel
  • Humans
  • Microchemistry
  • Peptide Hydrolases
  • Phosphopeptides
  • Protein Processing, Post-Translational
  • Substrate Specificity
  • Titanium

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