title: Highly selective enrichment of phosphopeptides by titanium (IV) attached monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate) microspheres.
abstract: A seeded polymerization protocol was developed for the synthesis of monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate), [poly(VPA-co-EDMA)] microspheres with superior porous properties. The protocol allowed the direct synthesis of phosphonic acid functionalized porous microspheres with the mean size of ∼4μm and the specific surface area of 420m2g-1without applying any complicated post-derivatization protocol for the attachment of phosphonic acid group. The phosphonic acid content of poly(VPA-co-EDMA) microspheres was determined as 1.5mmol H2PO3g-1microspheres. Ti(IV) ions were attached onto the microspheres via metal-chelate complex formation by phosphonate-groups and Ti(IV) carrying monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate), [Ti(IV)@poly(VPA-co-EDMA)] microspheres were obtained as a new sorbent for phosphopeptide enrichment via immobilized metal affinity chromatography. The phosphopeptides in the enriched samples were identified by matrix-assited laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Four different phosphopeptides were detected with extremely high intensity by the treatment of β-casein digest prepared with the concentration of 10 fmol/mL with Ti(IV)@poly(VPA-co-EDMA) microspheres. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts in a complex mixture of digested proteins (molar ratio of β-casein to BSA, 1:1000) and eight different phosphorylated peptides from BSA digest were successfully identified. Moreover, four highly intense signals of the phosphopeptides in human serum were observed with high S/N ratio and clear background after enrichment by using Ti(IV)@poly(VPA-co-EDMA) microspheres.
authors.affiliation: Chemical Engineering Department, Hacettepe University, 06800, Ankara, Turkey.
authors.forename: Kouroush
authors.initials: K
authors.lastname: Salimi
authors.affiliation: Department of Medical Biology and Genetics, Gazi University, 06500, Ankara, Turkey; Department of Medical Biology, Faculty of Medicine, İstanbul Medeniyet University, 34700, İstanbul, Turkey.
authors.forename: Duygu Deniz
authors.initials: DD
authors.lastname: Usta
authors.affiliation: Department of Chemistry, Hacettepe University, 06800, Ankara, Turkey; Institute of Science, Division of Bioengineering, Hacettepe University, 06800, Ankara, Turkey.
authors.forename: Ömür
authors.initials: Ö
authors.lastname: Çelikbıçak
authors.affiliation: Department of Medical Biochemistry, Faculty of Medicine, Hacettepe University, 06100, Ankara, Turkey.
authors.forename: Aslı
authors.initials: A
authors.lastname: Pınar
authors.affiliation: Department of Chemistry, Hacettepe University, 06800, Ankara, Turkey.
authors.forename: Bekir
authors.initials: B
authors.lastname: Salih
authors.affiliation: Chemical Engineering Department, Hacettepe University, 06800, Ankara, Turkey; Division of Nanotechnology and Nanomedicine, Hacettepe University, 06800, Ankara, Turkey. Electronic address: atuncel@hacettepe.edu.tr.
authors.forename: Ali
authors.initials: A
authors.lastname: Tuncel
mesh_terms: D002364:Caseins / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D008689:Methacrylates / Q000737:chemistry*; D008863:Microspheres; D010748:Phosphopeptides / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification*; D010757:Phosphorous Acids / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry; D058105:Polymerization; D011145:Polyvinyls / Q000737:chemistry*; D016062:Porosity; D014025:Titanium / Q000737:chemistry*
chemical_list: D002364:Caseins; D008689:Methacrylates; D010748:Phosphopeptides; D010757:Phosphorous Acids; D011145:Polyvinyls; C000618491:poly(vinylphosphonic acid-co-ethylene dimethacrylate); C570063:phosphonic acid; D014025:Titanium
keywords: Bovine serum albumin; Human serum; Monodisperse-porous microspheres; Phosphopeptide enrichment; Vinylphosphonic acid