Mass Spectrometric Analysis of Protein Mixtures at Low Levels Using Cleavable 13 C-ICAT and Multi-Dimensional Chromatography

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 multi-dimensional 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. cICAT labeled tryptic peptides were separated from non-labeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow LC-ESI MS/MS and LCMALDI MS/MS. 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.