title: Improving the selectivity of the phosphoric acid β-elimination on a biotinylated phosphopeptide.
abstract: This study aims at improving the MALDI-TOF detection of a phosphorylated peptide containing a cysteine residue by β-elimination of H(3)PO(4) hardly enriched by classical methods. The experimental conditions were optimized on this phosphopeptide (biot-pAdd) and its nonphosphorylated counterpart (biot-Add). The major side-reactions were H(2)S elimination on the cysteine residues and H(2)O elimination on the non phosphorylated serine residue of biot-Add. The former dilutes the MALDI-TOF signal for the desired species. The latter gives a product similar to what is obtained by H(3)PO(4) elimination and should prompt to caution when working with a mixture between phosphorylated and non phosphorylated peptides. Modifications on the solvent, the reaction temperature and time, the nature, and concentration of the base were made. Major improvement of the selectivity of the reaction was observed in 30 % ACN, at room temperature for 4 h. However, these optimizations are specific to these sequences and should be performed anew for different peptides. The selectivity of the reaction towards H(3)PO(4) elimination is improved, but the persistence of side-reactions renders a previous sample fractionation necessary. In these optimized conditions, the ionization enhancement is 3-fold and the detection limits for biot-pAdd are similar to biot-Add (100 fmol).
authors.affiliation: Laboratoire des Biomolécules, Université P et M Curie, UMR 7203 UPMC-CNRS-ENS, Paris, France.
authors.forename: Lucrèce
authors.initials: L
authors.lastname: Matheron
authors.forename: Séverine
authors.initials: S
authors.lastname: Clavier
authors.forename: Oumar
authors.initials: O
authors.lastname: Diebate
authors.forename: Philippe
authors.initials: P
authors.lastname: Karoyan
authors.forename: Gérard
authors.initials: G
authors.lastname: Bolbach
authors.forename: Dominique
authors.initials: D
authors.lastname: Guianvarc'h
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Sachon
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D006862:Hydrogen Sulfide / Q000737:chemistry; D057230:Limit of Detection; D010748:Phosphopeptides / Q000737:chemistry*; D010756:Phosphoric Acids / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature; D014867:Water / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D010748:Phosphopeptides; D010756:Phosphoric Acids; D014867:Water; D001710:Biotin; C030242:phosphoric acid; D003545:Cysteine; D006862:Hydrogen Sulfide; C032159:acetonitrile