Enrichment and separation of mono- and multiply phosphorylated peptides using sequential elution from IMAC prior to mass spectrometric analysis

Tine E Thingholm, Ole N Jensen, Martin R Larsen

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearch

Abstract

Phospho-proteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., immobilized metal affinity chromatography and titanium dioxide chromatography, provide varying degrees of selectivity and specificity for phosphopeptide enrichment. Furthermore, the number of multiply phosphorylated peptides that are identified in most published studies is rather low. Here the protocol for a new strategy that separates mono-phosphorylated pep-tides from multiply phosphorylated peptides using sequential elution from immobilized metal affinity chromatography is described. The two separate phosphopeptide fractions are subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multiply phosphorylated peptides, respectively, resulting in improved identification of especially multiply phosphorylated peptides from a minimum amount of starting material. The new method increases the coverage of the phosphoproteome significantly.
Original languageEnglish
Title of host publicationSpringer Protocols: Methods in Molecular Biology : Phospho-Proteomics: Methods and Protocols
EditorsMarjo de Graauw
Number of pages0
Place of PublicationUnited States
PublisherHumana Press
Publication date2009
Pages67-78, xi
DOIs
Publication statusPublished - 2009
SeriesMethods in Molecular Biology
Number527
ISSN1064-3745

Keywords

  • Algorithms
  • Animals
  • Chromatography, Affinity
  • Combinatorial Chemistry Techniques
  • Humans
  • Mass Spectrometry
  • Metals
  • Models, Biological
  • Phosphopeptides
  • Phosphorylation

Fingerprint Dive into the research topics of 'Enrichment and separation of mono- and multiply phosphorylated peptides using sequential elution from IMAC prior to mass spectrometric analysis'. Together they form a unique fingerprint.

  • Cite this

    Thingholm, T. E., Jensen, O. N., & Larsen, M. R. (2009). Enrichment and separation of mono- and multiply phosphorylated peptides using sequential elution from IMAC prior to mass spectrometric analysis. In M. D. Graauw (Ed.), Springer Protocols: Methods in Molecular Biology: Phospho-Proteomics: Methods and Protocols (pp. 67-78, xi). Humana Press. Methods in Molecular Biology, No. 527 https://doi.org/10.1007/978-1-60327-834-8_6