Tuesday, 7 December 2004
13:00 - 13:30

Automated proteome simplification and selective fractionation based on differential display

Guillaume Paton, Beckman-Coulter,  Roissy CDG, France

An ultimate result of proteomics is the understanding of complex biological systems, which can lead to new diagnostics and therapy. The first step toward this end is the discovery phase where protein differences between states of a proteome are profiled. One approach to proteome profiling fractionates the proteome into intact proteins with subsequent analysis for characterization and identification of the protein differences. This paper presents a multidimensional approach for proteome profiling that utilizes two-dimensional liquid chromatography for fractionation of the proteome followed by analysis with capillary electrophoresis (CE) and/or mass spectrometry (MS). The first-dimension separation is done by chromatofocusing, which separates proteins by pI. Fractions are collected based on pH intervals as detected by a pH monitor. These fractions are separated by hydrophobicity in a second dimension by high-resolution non porous silica reversed-phase chromatography. The proteins are detected by absorbance at 214 nm [1]. Fractions collected from the second dimension can be analyzed by CE for glycoproteins characterization in the human plasma proteome and MS for identification and intact mass measurement, allowing post-translational modification detection [2].

Different states of different types of proteomes were compared with this multidimensional fractionation and analysis approach, for identification of candidate cancer biomarkers [3] or for studies of modulation induced in human bladder cancer cell line by different extracellular matrix. Effect of abundant protein depletion on the human plasma proteome will be also illustrated.