OFFGEL-Isoelectric Focusing Fractionation for the Analysis of Complex Proteome

The proteomic analysis at the peptide level is increasingly becoming a method of choice for complex samples. The success, however, depends on the development of attractive peptide fractionation methodologies to decrease the sample complexity prior to mass analysis. Recently, the OFFGEL technology has emerged as being of great interest in shotgun proteomics. Using the Agilent 3100 OFFGEL fractionator, OFFGEL allows the in-solution separation of peptides from various biological sources by isoelectric focusing in up to highly resolved 24 fractions. In this chapter, we describe a detailed experimental protocol of a quantitative proteomic workflow including OFFGEL as a crucial step to improve the proteome �coverage. We provide the technical details concerning sample preparation, trypsin digestion, iTRAQ labelling, OFFGEL-IEF, RP nano-LC separation, MALDI-TOF/TOF MS/MS, and pI analysis. We rely on our recently published data to illustrate the use of OFFGEL as a powerful method for peptide �fractionation and as a filtering tool for pI -based validation of peptide identification.