Sequential elution from IMAC (SIMAC): An efficient method for enrichment and separation of mono-and multi-phosphorylated peptides

Tine E. Thingholm, Martin R. Larsen

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


Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the monophosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multiphosphorylated peptides from a minimum amount of starting material.

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


  • Immobilized metal affinity chromatography
  • Mass spectrometry
  • Multi-phosphorylated peptides
  • Phosphopeptide enrichment
  • Sequential elution
  • Titanium dioxide chromatography


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