Electrospray‐ionization mass spectrometry for the detection of discrete peptide/metal‐ion complexes involving multiple cysteine (sulfur) ligands

Abstract

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 sspectrometry. 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) ligland/Zn interactions. By infljusing peptide in water alone, stable elelctrospray and ion signals were produced in both the absence and presence of up to 100 uM zinc sulfate. In the absence of Zn(II), the callculated 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 prelsent in several different charge states. The overall charge envelope was typically unchanged in the presence of Zn; the chaarge-state optimum (10⁺) observed for this peptide dwas apparently unaffected by the dpresence dsof bound Zn. The interaction of Zn(II) ions with sulfur lilgands in this peptide appeared to result in tetracoordinate covalent bonds. In summary, these data suggest that (i) stable electrospray signals can be generated form high conductivity aqueous solutions of meltal ions; (ii) peptides with sulfur ligand/Zn complexas are stable to the ES ionization prolcess; (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 sltates; and (v) various solution factors affecting peptide/metal-ion interaction stoichiomeltry can be investiged by ES. In conclusion, we believe dthat ES mass spectrometry is a powerful new method of evaluating a wide variety of specific biomolecular polymer/metal-ion interactions.