Summary
Isotope-coded affinity tag (ICAT) labeling, in combination with mass spectrometry (MS), has been widely adopted as an effective method for comparing protein abundance levels. This chapter describes the ICAT labeling procedure in search for the celecoxib-regulated proteins in a colon cancer cell line. Celecoxib, a cyclooxygenase-2 (COX-2) specific inhibitor, is used as a colorectal cancer preventative drug in clinical trials. Here, celecoxib is used to inhibit the expression of COX-2 in a colon cancer cell line HT-29. To elucidate the proteomic changes induced by celecoxib, the protein lysates from the treated and control cells are prepared. The cysteine-containing proteins are labeled with the heavy and light ICAT reagents, respectively. The labeled proteins are then combined and digested with trypsin. The ICAT-labeled peptides are subject to the purification through an avidin column and eventually the cleavage of the biotin tags. This chapter focuses on the ICAT labeling procedure itself, because sample preparation is the most critical step of an ICAT-based protein expression comparison experiment. Other related procedures such as the cation exchange high performance liquid chromatography separation of peptides and MS analysis are detailed elsewhere in this book.
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Acknowledgments
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government.
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Xiao, Z., Veenstra, T.D. (2008). Comparison of Protein Expression by Isotope-Coded Affinity Tag Labeling. In: Vlahou, A. (eds) Clinical Proteomics. Methods in Molecular Biology™, vol 428. Humana Press. https://doi.org/10.1007/978-1-59745-117-8_10
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DOI: https://doi.org/10.1007/978-1-59745-117-8_10
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