Improved proteome coverage by using iTRAQ labelling and peptide OFFGEL fractionation.

The development of mass spectrometric techniques and fractionation methods now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Nevertheless, this work is particularly difficult due to the wide dynamic range of protein concentration in eukaryotic tissues. In this paper, we present a shotgun method whereby the peptides are fractionated using OFFGEL electrophoresis after iTRAQ labelling. We demonstrated that iTRAQ peptide labelling enhances MALDI ionisation and that the OFFGEL fractionation of the labelled peptides introduces a supplementary criterion (pI) useful for validation and identification of proteins. We showed that iTRAQ samples allowed lower-concentrated proteins identification in comparison with free-labelled samples. The combined use of iTRAQ labelling and OFFGEL fractionation allows a considerable increase in proteome coverage of very complex samples prepared from total cell extracts and supports the low-concentrated protein identification.