title: Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation.
abstract: Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large-scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large-scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two-step IMAC enrichment protocol under these conditions led to the identification of ∼100 phosphorylation sites from the tryptic digest of as little as 40 μL human serum.
authors.affiliation: Laboratory of Proteomics Research, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Eva
authors.initials: E
authors.lastname: Klement
authors.affiliation: Laboratory of Proteomics Research, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Timea
authors.initials: T
authors.lastname: Raffai
authors.affiliation: Laboratory of Proteomics Research, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.|Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D002846:Chromatography, Affinity / Q000379:methods*; D005290:Ferric Compounds / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010748:Phosphopeptides / Q000032:analysis* / Q000097:blood; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D059748:Proteolysis
chemical_list: D001798:Blood Proteins; D005290:Ferric Compounds; D010748:Phosphopeptides
keywords: Extracellular phosphorylation; Glycosylation; Human serum; IMAC; MS; Technology