title: Mimicking nature: Phosphopeptide enrichment using combinatorial libraries of affinity ligands.
abstract: Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins (α- and β-caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides.
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Iris L
authors.initials: IL
authors.lastname: Batalha
authors.affiliation: Cambridge Centre for Proteomics, Cambridge, CB2 1QR, UK.
authors.forename: Houjiang
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Cambridge Centre for Proteomics, Cambridge, CB2 1QR, UK.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Lilley
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, Cambridge, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Lowe
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Electronic address: cecilia.roque@fct.unl.pt.
authors.forename: Ana C A
authors.initials: AC
authors.lastname: Roque
mesh_terms: D000327:Adsorption; D000818:Animals; D040761:Biomimetic Materials; D002364:Caseins / Q000737:chemistry*; D002417:Cattle; D020650:Combinatorial Chemistry Techniques; D005290:Ferric Compounds; D008024:Ligands; D053758:Nanoparticles; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014227:Triazines / Q000737:chemistry
chemical_list: D002364:Caseins; D005290:Ferric Compounds; D008024:Ligands; D010748:Phosphopeptides; D014227:Triazines; C000499:ferric oxide; D012710:Serum Albumin, Bovine
keywords: Affinity; Iron oxide; Magnetic nanoparticles; Phosphopeptide enrichment; Phosphoproteomics; Synthetic ligands