Summary
The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+;, Ga3+, and Al3+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphopeptide enrichment by IMAC is sensititive and specific for peptide mixtures derived from pure proteins or simple protein mixtures. The selectivity of the IMAC method is, however, limited when working with peptide mixtures derived from highly complex samples, e.g., whole-cell extracts, where sample prefractionation is advisable. Furthermore, lowering the pH value of the sample loading buffer reduces nonspecific binding to the IMAC resin significantly, thereby improving the selectivity of IMAC for phosphopeptides. The retained phosphopeptides are released from the IMAC resin by using alkaline buffers (pH 10–11), EDTA, or phosphate-containing buffers. We have described a detailed and robust protocol for IMAC for phosphopeptide enrichment from semi-complex mixtures.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Chaga, G., Hopp, J., and Nelson, P. (1999) Immobilized metal ion affinity chromatogra-phy on Co2+ -carboxymethylaspartate–agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle Biotechnol Appl Biochem 29, 19–24.
Hochuli, E., Dobeli, H., and Schacher, A. (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues J Chromatogr 411, 177–84.
Andersson, L., and Porath, J. (1986) Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography Anal Biochem 154, 250–4.
Neville, D. C., Rozanas, C. R., Price, E. M., Gruis, D. B., Verkman, A. S., and Townsend, R. R. (1997) Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry Protein Sci 6, 2436–45.
Figeys, D., Gygi, S. P., McKinnon, G., and Aebersold, R. (1998) An integrated micro- fluidics-tandem mass spectrometry system for automated protein analysis Anal Chem 70, 3728–34.
Li, S., and Dass, C. (1999) Iron(III)-immo-bilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides Anal Biochem 270, 9–14.
Nuhse, T. S., Stensballe, A., Jensen, O. N., and Peck, S. C. (2003) Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatogra-phy and mass spectrometry Mol Cell Proteomics 2, 1234–43.
Posewitz, M. C., and Tempst, P. (1999) Immobilized gallium(III) affinity chromatography of phosphopeptides Anal Chem 71, 2883–92.
Stensballe, A., Andersen, S., and Jensen, O. N. (2001) Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis Proteomics 1, 207–22.
Ficarro, S. B., McCleland, M. L., Stukenberg, P. T., Burke, D. J., Ross, M. M., Shabanowitz, J., Hunt, D. F., and White, F. M. (2002) Phos- phoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae Nat Biotechnol 20, 301–5.
Gruhler, A., Olsen, J. V., Mohammed, S., Mortensen, P., Faergeman, N. J., Mann, M., and Jensen, O. N. (2005) Quantitative phosphopro-teomics applied to the yeast pheromone signaling pathway Mol Cell Proteomics 4, 310–27.
Stewart, I. I., Thomson, T., and Figeys, D. (2001) O-18 Labeling: a tool for proteomics Rapid Commun Mass Spectrom 15, 2456–65.
Krijgsveld, J., Gauci, S., Dormeyer, W., and Heck, A. (2006) In-gel isoelectric focusing of peptides as a tool for improved protein identification J Proteome Res 5, 1721–30.
Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J., Cohn, M. A., Cantley, L. C., and Gygi, S. P. (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins Proc Natl Acad Sci USA 101, 12130–5.
Saha, A., Saha, N., Ji, L. N., Zhao, J., Gre-gan, F., Sajadi, S. A. A., Song, B., and Sigel, H. (1996) Stability of metal ion complexes formed with methyl phosphate and hydrogen phosphate J Biol Inorg Chem 1, 231–38.
Kokubu, M., Ishihama, Y., Sato, T., Nagasu, T., and Oda, Y. (2005) Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis Anal Chem 77, 5144–54.
Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R., and Roepstorff, P. (1999) Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ioniza-tion mass spectrometry J Mass Spectrom 34, 105–16.
Liu, S., Zhang, C., Campbell, J. L., Zhang, H., Yeung, K. K., Han, V. K., and Lajoie, G. A. (2005) Formation of phosphopeptide-metal ion complexes in liquid chromatogra-phy/electrospray mass spectrometry and their influence on phosphopeptide detection Rapid Commun Mass Spectrom 19, 2747–56.
Jensen, S. S., and Larsen, M. R. (2007) Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom 21, 3635–45.
Acknowledgments
This work was supported by grants from the Danish Research Agency and the Lundbeck Foundation to O.N.J.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Thingholm, T.E., Jensen, O.N. (2009). Enrichment and Characterization of Phosphopeptides by Immobilized Metal Affinity Chromatography (IMAC) and Mass Spectrometry. In: Graauw, M.d. (eds) Phospho-Proteomics. Methods in Molecular Biology™, vol 527. Humana Press. https://doi.org/10.1007/978-1-60327-834-8_4
Download citation
DOI: https://doi.org/10.1007/978-1-60327-834-8_4
Publisher Name: Humana Press
Print ISBN: 978-1-60327-833-1
Online ISBN: 978-1-60327-834-8
eBook Packages: Springer Protocols