title: Evaluation of peptide/metal ion interactions by UV laser desorption time-of-flight mass spectrometry.
abstract: A relatively recent method developed to determine the molecular weights of intact peptides and proteins, matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), has been evaluated as a new means to investigate the metal ion-binding properties of model synthetic peptides. A contiguous sequence of 25 residues on the surface of the 74 kDa human plasma metal-binding transport protein histidine-rich glycoprotein (HRG) has been identified as a bioactive metal-binding domain. The peptide, (GHHPH)5G, was synthesized and evaluated by LDTOF-MS before and after the addition of Cu(II) in solution with 2,5-dihydroxybenzoic acid as the matrix. In the absence of added Cu(II), the major protonated molecular ion (M + H)+ was observed to have a mass equal to its calculated mass (2904.0 Da). In the presence of Cu(II), however, five additional peaks were observed at mass increments of approximately 63.9 Da. The maximum Cu(II)-binding capacity observed for the 26-residue peptide (5 g-atoms/mol) suggested that up to 1 Cu(II) may be bound per 5-residue internal repeat unit (GHHPH) within this peptide; several other monovalent and divalent metal cations were not bound under identical conditions of analysis. The Cu(II)-binding stoichiometry was verified by spectrophotometric titration and by frontal analyses of the immobilized peptide with a solution of Cu(II) ions. These results demonstrate the ability to verify directly the solution-phase binding capacity of metal-binding peptides by LDTOF-MS.
authors.affiliation: Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
authors.forename: T W
authors.initials: TW
authors.lastname: Hutchens
authors.forename: R W
authors.initials: RW
authors.lastname: Nelson
authors.forename: T T
authors.initials: TT
authors.lastname: Yip
mesh_terms: D000595:Amino Acid Sequence; D013058:Mass Spectrometry / Q000379:methods; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D008670:Metals; D010455:Peptides