Study of Metal Ion Labeling of the Conformational and Charge States of Lysozyme by Ion Mobility Mass Spectrometry

Abstract

The efficiency of Zn2+, Cu2+, Ni2+, Co2+, Fe2+ or Mn2+ labeling of the conformational and charge states of lysozyme was studied in H2O solvent at pH 2.5–6.8. Labeling of lysozyme was conducted with 50 M, 100 M and 500 M excess of the metal ion, resulting in the number of metal ions attached to lysozyme increasing two-fold over this range. At pH 6.2–6.8, Zn2+, Cu2+, Ni2+, Co2+ and Mn2+ labeled the highly folded 7+ conformer and the 8+ and 9+ partially unfolded conformers of lysozyme with the same number of metal ion tags, with only Fe2+ exhibiting no labeling. Lysozyme conserved its charge after metal ion labeling which shows at each charge state the divalent metal ion is replacing two protons. As the pH is lowered to 4.7–5.0 and 2.5–2.9, the labeling of lysozyme by Zn2+, Cu2+, Ni2+, Co2+ or Mn2+ decreased in efficiency due to increased competition from protons for the aspartate and glutamate binding sites. The metal ions preferentially labeled the highly folded 7+ and partially unfolded 8+ conformers, but labeling decreased as the charge of lysozyme increased. In contrast to the other metal ions, Fe2+ exhibited labeling of lysozyme only at the lowest pH of 2.8. At higher pH, the oxidation of Fe2+ and formation of hydroxy-bridged complexes probably make the Fe2+ unreactive towards lysozyme.