Comparison of protein expression by isotope-coded affinity tag labeling.

摘要: 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 search celecoxib-regulated proteins a colon cancer cell line. Celecoxib, cyclooxygenase-2 (COX-2) specific inhibitor, is used colorectal preventative drug clinical trials. Here, celecoxib to inhibit expression of COX-2 line HT-29. To elucidate proteomic changes induced by celecoxib, lysates from treated and control cells are prepared. The cysteine-containing labeled heavy light reagents, respectively. then combined digested trypsin. ICAT-labeled peptides subject purification through avidin column eventually cleavage biotin tags. focuses on itself, because sample preparation most critical step ICAT-based comparison experiment. Other related procedures such cation exchange high performance liquid chromatography separation MS analysis detailed elsewhere this book.