OFFGEL-Isoelectric Focusing Fractionation for the Analysis of Complex Proteome

  • Emilie Ernoult
  • Catherine GuetteEmail author
Part of the Neuromethods book series (NM, volume 57)


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.

Key words

OFFGEL Peptide fractionation Preparative IEF Shotgun proteomics Proteome coverage iTRAQ labelling Biomarker discovery Methodology 


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Laboratory of Oncopharmacology-PharmacogeneticsCentre INSERM Régional de Recherche sur le Cancer U892, Centre Régional de Lutte Contre le Cancer Paul PapinAngersFrance

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