(424e) Fractionation of Complex Peptide Mixtures by IEF: Comparison of off-Gel with In-Gel Separations

Shotgun proteomic analysis involves the separation of proteolytically digested samples by reverse-phase liquid chromatography followed by mass spectrometry with electrospray ionization (LC-MS). This technique can detect thousands of individual peptides while identifying their protein of origin. It thus provides a powerful profiling technique for proteins present in a biological sample. Coupling this technique with an upstream, orthogonal separation technique yielding several fractions to be analyzed separately results in higher total numbers of identified peptides greater depth of proteome coverage. Isoelectric focusing (IEF) in an immobilized pH gradient (IPG) is a common upstream fractionation method used for this purpose. The IEF separation may be performed “Off-gel” in which the digest is electrophoretically fractionated through a series of sample cups placed in contact with an IPG gel (1), or “In-gel” in which the digest is incorporated into an IPG gel by rehydration and separated within the gel matrix (2). Off-gel separated peptides are recovered directly from the sample cups, while in-gel separated peptides are eluted from excised sections of IPG gel. This study was undertaken in order to compare the two methods in terms of overall speed and simplicity of the workflow, depth of proteome coverage provided, and the resolution of the separation as judged by the percentage of peptides restricted to a single fraction.

The resolution of the two different separation techniques was similar; however the in-gel separation resulted in approximately 20% more peptides detected. The in-gel separation was more rapid, but required additional time for peptide elution following the separation. An additional advantage of the in-gel separation is greater flexibility in determining the pH range covered by each individual fraction. In-gel peptide IEF fractionation can be performed with relatively inexpensive equipment and offers distinct benefits compared to off-gel peptide IEF fractionation.

(1) Hörth, P. et al. (2006). Molecular and Cellular Proteomics 5:1968-1974

(2) Cargile, B.J et al. (2005). Journal of Biomolecular Techniques 16:181-189