Phosphoproteomics by Highly Selective IMAC Protocol
Protein phosphorylation plays an important role in biological process such as cell differentiation, cell cycle control, metabolism, and apoptosis. Toward global analysis of the phosphoproteome, enrichment is an essential step to overcome analytical challenges associated with the nature of phosphoprotein, including their dynamic modification patterns, substoichiometric concentrations, heterogeneous forms of phosphoproteins, and low mass spectrometric response. Here, based on detailed evaluation of the capture and release mechanism in immobilized metal affinity chromatography (IMAC), we provide a pH/acid-controlled IMAC protocol for phosphopeptide purification with high specificity and lower sample loss. Based on a model study on non-small-cell lung cancer cell, better than 90% phosphopeptide enrichment specificity can be achieved without the use of commonly adapted methyl esterification procedure. In addition, the protocol is compatible to fractionation using SDS-PAGE. We have successfully employed the pH/acid-controlled IMAC enrichment strategy to characterize over 2,360 nondegenerate phosphopeptides and 2,747 phosphorylation sites in H1299 lung cancer cell line. We expect that the simple and reproducible IMAC protocol can be applied, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome associated with neurodegenerative diseases.
Key wordsIMAC SDS-PAGE Phosphoproteomics Mass spectrometry
This work was supported by Academia Sinica and the National Science Council in Taiwan. We thank Dr. Jeou-Yuan Chen for providing human non-small cell lung carcinoma cell line (H1299).
- 7.David, C. A. N., Townsend, R. R., Christine, R. R., Verkman, A. S., Elmer, M. P., and Darren, B. G. (1997) Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry, Protein Science 6, 2436–2445.Google Scholar
- 10.Corthals, G. L., Aebersold, R., Goodlett, D. R., and Burlingame, A. L. (2005) Identification of Phosphorylation Sites Using Microimmobilized Metal Affinity Chromatography, in Abelson, J. N., Simon, M. I., Colowick, S. P., Kaplan, N. O. (eds.) Methods in Enzymology, pp 66–81, Academic Press.Google Scholar
- 14.Salomon, A. R., Ficarro, S. B., Brill, L. M., Brinker, A., Phung, Q. T., Ericson, C., Sauer, K., Brock, A., Horn, D. M., Schultz, P. G., and Peters, E. C. (2003) Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry, Proceedings of the National Academy of Sciences of the United States of America 100, 443–448.PubMedCrossRefGoogle Scholar
- 21.Tsai, C.-F., Wang, Y.-T., Chen, Y.-R., Lai, C.-Y., Lin, P.-Y., Pan, K.-T., Chen, J.-Y., Khoo, K.-H., and Chen, Y.-J. (2008) Immobilized Metal Affinity Chromatography Revisited: pH/Acid Control toward High Selectivity in Phosphoproteomics, Journal of Proteome Research 7, 4058–4069.PubMedCrossRefGoogle Scholar
- 22.Han, C.-L., Chien, C.-W., Chen, W.-C., Chen, Y.-R., Wu, C.-P., Li, H., and Chen, Y.-J. (2008) A Multiplexed Quantitative Strategy for Membrane Proteomics: Opportunities for Mining Therapeutic Targets for Autosomal Dominant Polycystic Kidney Disease, Mol Cell Proteomics 7, 1983–1997.PubMedCrossRefGoogle Scholar