Comparison of Phosphoproteomic Separation Strategies Based on Strong Cation Exchange Chromatography and Isoelectric Focusing Techniques

Efficient pre-purification steps for the enrichment of phosphorylated proteins or phosphopeptides are necessary for the detection of phosphorylation sites in phosphoproteomic analysis. Currently, the most common first-dimensional separation technique is strong cation exchange (SCX). A potential alternative to SCX-based separation is to use isoelectric focusing (IEF) as a first-dimensional separation technique, which has been demonstrated recently. In this article, we present a direct comparison between SCX and IEF based on IPG strips (IPG-IEF) for phosphoproteomic separation. The comparison experiments were carried out using standard phosphoproteins and a real sample of HepG2 cell. Then, the comparison of 18O labeling phosphopeptides' stability under IPG-IEF with that under SCX was made. Fractions obtained from both the techniques (SCX and IPG-IEF) were analyzed using High Mass Accuracy LTQ-FTICR-MS/MS. The results demonstrated that SCX and IPG-IEF are useful in phosphopeptides enrichment analyses on a large scale and that SCX is superior to IPG-IEF, whereas the 18O labeling phosphopeptides' stability under SCX is poor compared with that under IPG-IEF.