Complementary workflow for global phosphoproteome analysis.

We described a workflow involving a combination of titanium dioxide (TiO(2)) enrichment, strong anion exchange (SAX), and strong cation exchange (SCX) fractionation for global phosphoproteome analysis. The workflow proposed TiO(2) -based high efficient enrichment with optimum peptide-to-beads ratio prior to robust IEC fractionation. With the optimum peptide-to-beads ratio, offline TiO(2) enrichment provides high selectivity and large sample loading capacity compared with online TiO(2) chromatography. The eluate with highly enriched phosphopeptides is then subjected to online SAX and SCX fractionation coupled to RP-LC-MS/MS analysis. The identification of phosphopeptides from SAX, SCX, and flow-through fractions showed high complementary features. Importantly, large amount of multiphosphopeptides could be recovered in SAX fractionations. In total, up to 5063 unique phosphosites were identified from 4557 unique phosphopeptides using 4-mg HeLa cell lysate as the starting material.