Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling

Jérôme Chenau¹ , Sylvie Michelland¹^,3 , Jonathan Sidibe² and Michel Seve¹^,3

¹Centre de Recherche INSERM/UJF U823, Cibles Diagnostiques ou Thérapeutiques et Vectorisation des Drogues dans le Cancer du Poumon Institut Albert Bonniot, 38706 La Tronche, France

²Université Joseph Fourier, 38041 Grenoble Cedex 09, France

³Centre d'Innovation en Biologie, Pavillon B, CHU de la Tronche, 38043 Grenoble Cedex 9, France

author email corresponding author email

Proteome Science 2008, 6:9doi:10.1186/1477-5956-6-9


Published:	26 February 2008

Abstract

Background

The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes.

Results

We evaluated OFFGEL fractionator technology to separate peptides from two complex proteomes, human secretome and human plasma, using a 24-wells device encompassing the pH range 3–10. In combination with reverse phase liquid chromatography, peptides from these two samples were separated and identified by MALDI TOF-TOF. The repartition profiles of the peptides in the different fractions were analyzed and explained by their content in charged amino acids using an algorithmic model based on the possible combinations of amino acids. We also demonstrated for the first time the compatibility of OFFGEL separation technology with the quantitative proteomic labeling technique iTRAQ allowing inclusion of this technique in complex samples comparative proteomic workflow.

Conclusion

The reported data showed that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic proteomes (plasma and secretome) and is compatible with iTRAQ labeling quantitative studies. We therefore consider peptides OFFGEL fractionation as an effective addition to our strategy and an important system for quantitative proteomics studies.