Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable 13C-Isotope-coded Affinity Tag and Multidimensional Chromatography

• DOI: 10.1074/mcp.m300021-mcp200
• Publication Date: 2003-05-27T00:16:28Z
• Authors: Kirk C. Hansen; Gerold Schmitt-Ulms; Robert J. Chalkley; Jan Hirsch; Michael A. Baldwin; A.L. Burlingame
• Keywords: Isotope Analysis

In order to identify and compare the protein content of very low quantity samples high complexity, a protocol has been established that combines differential profiling strength new cleavable 13C isotope-coded affinity tag (cICAT) reagent with sequence coverage provided by multidimensional liquid chromatography two modes tandem mass spectrometry. Major objectives during optimization were minimize sample losses establish robust procedure employs volatile buffer systems are highly compatible Cleavable ICAT-labeled tryptic peptides separated from nonlabeled avidin chromatography. Subsequently, peptide analyzed nanoflow electrospray ionization spectrometry matrix-assisted laser desorption/ionization The use ionization/instrumental configurations led complementary identifications increased confidence assignments. Examples illustrate power this strategy taken different projects: i) immunoaffinity purified complexes containing prion murine brain, ii) human tracheal epithelium gland secretions. these studies, large number novel proteins identified using stringent match criteria, in addition many had previous experiments. latter case, ICAT method produced significant information on changes occur expression levels patient suffering cystic fibrosis.