Isolation of phosphopeptides by p/-difference-based electrophoresis

Effective proteomics studies of protein phosphorylation require an efficient enrichment method for phosphopeptides, which remains a challenge. Here, we describe the discovery of p/differences between methylated phosphopeptides (typically <7.4) and methylated nonphosphorylated peptides (typically >9.0). This p/ difference allows isolation of methylated phosphopeptides from the methylated nonphosphopeptides by in-solution isoelectric focusing. We proved the principle of such a novel approach by isolating a phosphorylated peptide from nonphosphorylated tryptic digest of myoglobin. While the principle for p/-based, in-solution electrophoresis is proven, it requires further development for practical application. The method described here provides a stepping stone toward more reliable, convenient method for efficient isolation of phosphopeptides.