Phosphopeptide enrichment by immobilized metal affinity chromatography

Tine E. Thingholm, Martin R. Larsen

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

Abstract

Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of nonphosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10–11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe 3+ for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

Original languageEnglish
Title of host publicationPhospho-Proteomics : Methods and Protocols
EditorsLouise von Stechow
PublisherHumana Press
Publication date2016
Edition2.
Pages123-133
Chapter8
ISBN (Print)978-1-4939-3048-7
ISBN (Electronic)978-1-4939-3049-4
DOIs
Publication statusPublished - 2016
SeriesMethods in Molecular Biology
Volume1355
ISSN1064-3745

Keywords

  • Immobilized metal affinity chromatography
  • Mass spectrometry
  • Phosphopeptide enrichment
  • Protein phosphorylation

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