title: Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography.
abstract: The mixture of phosphopeptides enriched from proteome samples are very complex. To reduce the complexity it is necessary to fractionate the phosphopeptides. However, conventional enrichment methods typically only enrich phosphopeptides but not fractionate phosphopeptides. In this study, the application of strong anion exchange (SAX) chromatography for enrichment and fractionation of phosphopeptides was presented. It was found that phosphopeptides were highly enriched by SAX and majority of unmodified peptides did not bind onto SAX. Compared with Fe(3+) immobilized metal affinity chromatography (Fe(3+)-IMAC), almost double phosphopeptides were identified from the same sample when only one fraction was generated by SAX. SAX and Fe(3+)-IMAC showed the complementarity in enrichment and identification of phosphopeptides. It was also demonstrated that SAX have the ability to fractionate phosphopeptides under gradient elution based on their different interaction with SAX adsorbent. SAX was further applied to enrich and fractionate phosphopeptides in tryptic digest of proteins extracted from human liver tissue adjacent to tumorous region for phosphoproteome profiling. This resulted in the highly confident identification of 274 phosphorylation sites from 305 unique phosphopeptides corresponding to 168 proteins at false discovery rate (FDR) of 0.96%.
authors.affiliation: National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, China.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Han
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Houjiang
authors.initials: H
authors.lastname: Zhou
authors.forename: Xinning
authors.initials: X
authors.lastname: Jiang
authors.forename: Shun
authors.initials: S
authors.lastname: Feng
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Jiang
authors.forename: Ruijun
authors.initials: R
authors.lastname: Tian
authors.forename: Dafang
authors.initials: D
authors.lastname: Wan
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
authors.forename: Jianren
authors.initials: J
authors.lastname: Gu
mesh_terms: D000595:Amino Acid Sequence; D005591:Chemical Fractionation / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002853:Chromatography, Liquid; D006801:Humans; D008099:Liver / Q000737:chemistry*; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D020543:Proteome