Off-line high pH reversed-phase fractionation for in-depth phosphoproteomics

In this study we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC-MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in total number of phosphopeptides detected when analyzing the same number of fractions by identical 70 minutes LC-MS/MS gradients