The kinetic method is applied to differentiate and quantify mixtures of isomeric tripeptides based on the competitive dissociations of divalent metal ion-bound clusters in an ion trap mass spectrometer. This methodology is extended further to determine compositions of ternary mixtures of the isomers Gly-Gly-Ala (GGA), Ala-Gly-Gly (AGG), and Gly-Ala-Gly (GAG). This procedure also allows to perform chiral quantification of a ternary mixture of optical isomers. The divalent metal ion Ca II is particularly appropriate for isomeric distinction and quantification of the isobaric tripeptides Gly-Gly-Leu/Gly-Gly-Ile (GGL/GGI). Among the first-row transition metal ions, Cu II yields remarkably effective isomeric differentiation for both the isobaric tripeptides, GGI/GGL using GAG as the reference ligand, and the positional isomers GAG/GGA using GGI as the reference ligand. This is probably due to agostic bonding: âŁ-agostic bonding occurs between Cu II and GAG and â€-agostic bonding between Cu II and GGI, each produces large but different steric effects on the stability of the Cu II -bound dimeric clusters. These data form the basis for possible future quantitative analyses of mixtures of larger peptides such as are generated, for example, in combinatorial synthesis of peptides and peptide mimics. . Some metallopeptides recognize and interact selectively with the DNA minor groove as a function of the identity, chirality, and positioning of amino acid side chains within the peptide-ligand framework [2]. The conformational changes induced by metal-ion binding can be immense and remarkable [3]. The effective positioning of functional groups or appropriate subunits within the peptides allows them to trigger new functions [4]. In addition, the modular synthetic strategies used in polypeptides are prone to combinatorial selection. As a result of all these considerations, peptide research as it relates to drug discovery and design has become an important field with the potential to generate new drugs [5]. Direct tests of the purity of combinatorial mixtures, especially those containing isomeric peptides that are not always easily delineated, can provide information about the reliability of synthetic protocols [6]. Therefore, simple, fast and sensitive methods are highly desirable to determine isomeric peptide contamination [7]. This paper reports on steps to develop mass spectrometric procedures for this purpose.The emergence of soft ionization techniques such as electrospray ionization (ESI) [8] and matrix-assisted laser desorption ionization (MALDI) [9] has made mass spectrometry a major method for peptide characterization [10]. Distinction between leucine and isoleucine residues can be achieved by collision-induced dissociation (CID) [11], for example in the case of the isomeric tripeptides Gly-XXX-Arg, by studying side-chain radical losses from radical cations in a tandem mass spectrometric experiment [12]. Gas-phase calcium ion binding (including sites and affinities) in various peptides has been studied using both low-energ...