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)~5~G, 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) suggest that up 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 LDTOC‐MS.