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Molecular & Cellular Proteomics 2:299-314, 2003.
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

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

Kirk C. Hansen^,, Gerold Schmitt-Ulms^¶, Robert J. Chalkley, Jan Hirsch^||, Michael A. Baldwin^,¶ and A. L. Burlingame^,¶,**

Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, University of California San Francisco, San Francisco, California 94143-0446
^¶ Institute for Neurodegenerative Diseases, University of California San Francisco, San Francisco, California 94143
^|| Cardiovascular Research Institute, University of California San Francisco, California 94143-0130

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

^** To whom correspondence should be addressed. E-mail: alb{at}itsa.ucsf.edu

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