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Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography

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Phospho-Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1355))

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 non-phosphorylated 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 Fe3+ for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

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Acknowledgements

This work was supported by the Danish Natural Science and Medical Research Councils (grant no. 10-082195 (T.E.T.)) and the Lundbeck Foundation (M.R.L.—Junior Group Leader Fellowship).

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Authors and Affiliations

  1. Division of Translational Cancer Research, Lund University, Lund, Sweden

    Tine E. Thingholm

  2. Lund Stem Cell Center, Lund University, Lund, Sweden

    Tine E. Thingholm

  3. Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark

    Martin R. Larsen

Authors
  1. Tine E. Thingholm
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  2. Martin R. Larsen
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Corresponding author

Correspondence to Martin R. Larsen .

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Editors and Affiliations

  1. University of Copenhagen, Center for Protein Research, Copenhagen Nm, Denmark

    Louise von Stechow

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© 2016 Springer Science+Business Media New York

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Thingholm, T.E., Larsen, M.R. (2016). Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography. In: von Stechow, L. (eds) Phospho-Proteomics. Methods in Molecular Biology, vol 1355. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-3049-4_8

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  • DOI: https://doi.org/10.1007/978-1-4939-3049-4_8

  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-3048-7

  • Online ISBN: 978-1-4939-3049-4

  • eBook Packages: Springer Protocols

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