title: Complementary workflow for global phosphoproteome analysis.
abstract: 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.
authors.affiliation: Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Qing-Run
authors.initials: QR
authors.lastname: Li
authors.forename: Zhi-Bin
authors.initials: ZB
authors.lastname: Ning
authors.forename: Xing-Lin
authors.initials: XL
authors.lastname: Yang
authors.forename: Jia-Rui
authors.initials: JR
authors.lastname: Wu
authors.forename: Rong
authors.initials: R
authors.lastname: Zeng
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D010748:Phosphopeptides / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010748:Phosphopeptides