title: Design and optimization of a phosphopeptide anchor for specific immobilization of a capture protein on zirconium phosphonate modified supports.
abstract: The attachment of affinity proteins onto zirconium phosphonate coated glass slides was investigated by fusing a short phosphorylated peptide sequence at one extremity to enable selective bonding to the active surface via the formation of zirconium phosphate coordinate covalent bonds. In a model study, the binding of short peptides containing zero to four phosphorylated serine units and a biotin end-group was assessed by surface plasmon resonance-enhanced ellipsometry (SPREE) as well as in a microarray format using fluorescence detection of AlexaFluor 647-labeled streptavidin. Significant binding to the zirconated surface was only observed in the case of the phosphopeptides, with the best performance, as judged by streptavidin capture, observed for peptides with three or four phosphorylation sites and when spotted at pH 3. When fusing similar phosphopeptide tags to the affinity protein, the presence of four phosphate groups in the tag allows efficient immobilization of the proteins and efficient capture of their target.
authors.affiliation: Department of Chemistry, University of Florida , Gainesville, Florida 32611-7200, United States.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.forename: Clémence
authors.initials: C
authors.lastname: Queffélec
authors.forename: Cathy
authors.initials: C
authors.lastname: Charlier
authors.forename: Alain
authors.initials: A
authors.lastname: Defontaine
authors.forename: Amina
authors.initials: A
authors.lastname: Fateh
authors.forename: Charles
authors.initials: C
authors.lastname: Tellier
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Talham
authors.forename: Bruno
authors.initials: B
authors.lastname: Bujoli
mesh_terms: D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D040081:Protein Array Analysis / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry*; D015040:Zirconium / Q000737:chemistry*
chemical_list: D055767:Immobilized Proteins; D010455:Peptides; C027006:zirconium phosphate; D019809:Streptavidin; D015040:Zirconium