We extended the kinetic method to determine the intrinsic affinities of nonvolatile organic molecules with divalent metal ions and then applied the amended method to determine the calcium affinities of peptide analogs of the calcium-binding site III of rabbit skeletal troponin C. Metal-bis(peptide) complexes of the composition ([H2Pi + H2Pii] - H + Ca)+, where H2P is a neutral peptide, were introduced into the gas phase by fast atom bombardment. The extended kinetic method recognizes that the dissociation characteristics of a singly charged, bis(peptide) complexes of divalent metal ions are determined by not only the metal-ion affinity but also the proton affinities of the neutral and deprotonated peptides. The modified method requires one to measure the relative abundances of [H2P - H + Ca]+, [H2P + H]+, and [H2P - H]- ions that form upon collisional activation of mixed peptide/metal complexes, proton-bound peptide dimers, and deprotonated peptide dimers, respectively. We found, by using the modified method, that the set of peptides has a different affinity order than that in solution. Peptides with one aspartic acid have a higher intrinsic Ca2+ affinity than those with two aspartates. The location of the aspartic acid (Asp) residues at various positions also affects the Ca2+ affinity. Those peptides with one Asp in the middle of the chain have higher Ca2+ affinities than those with Asp on the end because the former peptides offer greater polarizability to stabilize the charge. Peptides with two Asp's located in close proximity have higher intrinsic calcium affinities than those with aspartates positioned further apart.