BBB seminar: Joël Vandekerckhove
Novel Concepts and Strategies Leading to Second Generation Broad Spectrum Non-Gel Proteomics
Department of Biochemistry,Faculty of Medicine and Health Sciences, VIB, Ghent University, Ghent, Belgium
An increasing number of genomic sequences are known today and scientists are now trying to understand gene functions. This next step is largely addressed by proteomics, covering large scale protein analysis, taking the genome sequences as a starting point. This involves protein expression profiling, analysis of post-translational modifications, protein processing, protein-protein interactions, sub-cellular localisation etc… Such integrated studies clearly represent a number of huge technical challenges, where only combinations of different technologies may lead to solutions and insights.
The novel methodology presented here is based on an analysis of peptides obtained by digesting the proteins present in total cell lysates. Out of this highly complex mixture we select a subset of peptides, thereby reducing the complexity, while keeping the representativity for the parent proteins. This sorting procedure is based on the concept of 'diagonal chromatography' and has been adapted for peptide mixtures containing up to 100,000 components. COFRADIC™ (COmbined FRActional DIagonal Chromatography) allows specific sorting of either methionine or cysteine containing peptides. It also allows the isolation of peptides from the amino terminal ends of proteins, thereby providing means to study limited protein degradation or processing and the identification of in vivo acetylated proteins. Recently, we were successful in sorting for phospho-peptides in total cell extracts. Each of these sorting protocols can be carried out by the same chromatographic system, using simple chemistries adapted for each question. Sorted peptides are identified by high-throughput mass spectrometry and database searching using novel algorithms. The entire process including sorting and identification can be carried out in a fully automated manner in less than one day.
Illustrations will be shown using cytoskeletal preparations of human blood platelets revealing novel data which will form the basis for follow-up by future functional analyses.
References: Gevaert et al. Mol. Cell. Prot.1, 896-903,(2002); Gevaert et al. Nat. Biotechnol. 21, 566-569.(2003).
Prof. Vandekerckhove's main research activity consists in developing novel technologies based upon mass spectrometry in proteome research. The central research line is the development of quantitative gel-free proteomics, a novel concept that is particularly adapted for automated high-throughput analysis with sensitivity outperforming conventional 2D-gel systems. These techniques can be adopted for analysis of post-translational modifications as well as large-scale proteome projects. He has also developed a novel technique for the study of interactions between components of mammalian signalling systems, and novel methods for the study of the cell cytoskeleton.