ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY FOR THE DETECTION OF DISCRETE PEPTIDE METAL-ION COMPLEXES INVOLVING MULTIPLE CYSTEINE (SULFUR) LIGANDS

Conditions have been developed to characterize the reversible interaction of one or more Zn(II) ions with cysteine (sulfur) ligands on metal-binding peptides by electrospray-ionization (ES) mass spectrometry. A 71-residue peptide with two separate clusters of four cysteine residues was selected as a model to optimize both the solution and electrospray variables most likely to affect the detection of stable cysteine (sulfur) ligand/Zn interactions. By infusing peptide in water alone, stable electrospray and ion signals were produced in both the absence and presence of up to 100-mu-M zinc sulfate. In the absence of Zn(II), the calculated mass of the fully reduced peptide (8248.5 Da) was observed (8248.4 +/- 0.4 Da). In the presence of Zn(II), peptides with zero, one and two bound Zn atoms were detected; all three species were present in several different charge states. The overall charge envelope was typically unchanged in the presence of Zn; the charge-state optimum (10+) observed for this peptide was apparently unaffected by the presence of bound Zn. The interaction of Zn(II) ions with sulfur ligands in this peptide appeared to result in tetracoordinate covalent bonds. In summary, these data suggest that (i) stable electrospray signals can be generated from high conductivity aqueous solutions of metal ions; (ii) peptides with sulfur ligand/Zn complexes are stable to the ES ionization process; (iii) bound Zn is not the primary source of charge and does not alter the observed charge-envelope optimum; (iv) the relative distribution of peptide without bound Zn, with one bound Zn, and with two bound Zn atoms can be fully resolved in each of several different charge states; and (v) various solution factors affecting peptide/metal-ion interaction stoichiometry can be investigated by ES. In conclusion, we believe that ES mass spectrometry is a powerful new method of evaluating a wide variety of specific biomolecular polymer/metal-ion interactions.