Reversed-phase-reversed-phase liquid chromatography approach with high orthogonality for multidimensional separation of phosphopeptides.

Protein phosphorylation regulates a series of important biological processes in eukaryotes. However, the phosphorylation sites found up to now are far below than that actually exists in proteins due to the extreme complexity of the proteome sample. Here a new reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach was developed for multidimensional separation of phosphopeptides. In this approach, a large number of fractions were collected from the first dimensional RPLC separation at high pH. And then these fractions were pooled every two fractions with equal time interval, one from the early eluted section and another one from the later eluted section. The pooled fractions were finally submitted to RPLC-tandem mass spectrometry (MS/MS) analysis at low pH. It was found the resulting 2D separation was highly orthogonal and yielded more than 30% phosphopeptide identifications over the conventional RP-RPLC approach. This study provides a powerful approach for efficient separation of phosphopeptides and global phosphorylation analysis, where the orthogonality of 2D separation is greatly improved and the first dimensional separation is of high resolution.