title: Sequence-dependent enrichment of a model phosphopeptide: a combined MALDI-TOF and NMR study.
abstract: The goal of this study was to detect and quantify by MALDI-TOF MS the phosphorylation of a peptide containing the recognition motif of the Protein Kinase C (PKC). Such model peptide can be used as a phosphorylation probe to follow intracellular kinase/phosphatase activities. This study allowed us to establish relationships between sequence specificities and affinity for TiO(2) or IMAC media. The peptide has the sequence biotin-GGGGCFRTPSFLKK-NH(2) in which the serine residue can be phosphorylated. Enrichment of the corresponding phosphopeptide, by the dedicated IMAC and TiO(2) affinity chromatography methods, proved inefficient. By combining MALDI-TOF and NMR data, we first showed that the lack of affinity of the phosphopeptide for TiO(2) was partly related to the basic property of its peptide sequence. Furthermore, the peptide shows local structuration around the P(9)- S(10) segment, with formation of a salt bridge between the guanidinium group of the R(7) side chain and the phosphate moiety. In conjunction with an inadequate position of the {biotin-G(4)} N-terminal tag, this local structure could shield the phosphate group, preventing interaction with TiO(2). To improve TiO(2) affinity, the peptide sequence was modified accordingly. The new sequences retained the biological properties while their enrichment by IMAC or TiO(2) became possible.
authors.affiliation: Université P. et M. Curie Paris 6, UMR 7203 CNRS-UPMC-ENS, 4, Place Jussieu, 75005 Paris, Ecole Normale Supérieure, Département de Chimie, 24, rue Lhomond, 75005 Paris, France.
authors.forename: Lucrèce
authors.initials: L
authors.lastname: Matheron
authors.forename: Matheron
authors.initials: M
authors.lastname: Lucrèce
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Sachon
authors.forename: Sachon
authors.initials: S
authors.lastname: Emmanuelle
authors.forename: Fabienne
authors.initials: F
authors.lastname: Burlina
authors.forename: Burlina
authors.initials: B
authors.lastname: Fabienne
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
authors.forename: Sagan
authors.initials: S
authors.lastname: Sandrine
authors.forename: Olivier
authors.initials: O
authors.lastname: Lequin
authors.forename: Lequin
authors.initials: L
authors.lastname: Olivier
authors.forename: Gèrard
authors.initials: G
authors.lastname: Bolbach
authors.forename: Bolbach
authors.initials: B
authors.lastname: Gèrard
mesh_terms: D000595:Amino Acid Sequence; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010748:Phosphopeptides / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010748:Phosphopeptides; D011493:Protein Kinase C