Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometry

Abstract

A method has been developed that combines electrospray ionization mass spectrometry with pH control to provide analysis of metals in native or reconstituted metallothioneins. These metalloproteins cooperatively bind seven divalent metal ions, most commonly Zn2+ and Cd2+. Since the protein is denatured and metal ions are lost below pH3, the pH of the electrospray solution is critical to successful results. The metal-free apoprotein was detected with its most abundant ions in a charge state of 6+, while the folded metallothionein-metal complexes were observed with lower charge states. The retention of seven metals in the molecular ions detected is consistent with the hypothesis that metallothionein retains its conformation in the gas phase. This mass spectrometric technique can be used to determine rapidly and accurately how many and what cations are incorporated per molecule of protein. Information about molar distributions and estimates of relative abundances of various complexes in the sample can be acquired in a single measurement.

Monitoring metal ion flux in reactions of metallothionein and drug‐modified metallothionein by electrospray mass spectrometry

“…The electrospray spectrum of purified thionein 2a and 2a' in Figure 5A shows the presence of small amounts of Znl and Zn4 species. Fragmentation, for example, loss of the acetyl group and loss of acetylmethionine from the N-terminus in a small percentage of the protonated apo and holo protein molecules, has been reported previously (Yu et al, 1993), and results from the electrospray process. This is the source of the small peaks at masses just below that of thionein 2a.…”

“…Fully metallated MT is detected in aqueous solution at pH 7.5 using electrospray mass spectrometry, with the 4+ charge state by far the most abundant (Yu et al, 1993). Partially metallated MT species also produce predominantly the 4+ charge state in these on-line experiments.…”

“…Electrospray studies of zinc-and cadmium-containing metallothionein were initiated in this laboratory with experiments at several pH values, and by comparison of the metal ion determinations with parallel analyses made using graphite furnace atomic absorption (Yu et al, 1993). Care was taken in all the experiments reported here to exclude artifactual clustering by using low concentration, aqueous solutions, and spray conditions that provide steady ion currents.…”

“…The relation of the electrospray spectrum to the composition of the aqueous solution has been tested previously by comparison to atomic absorption measurements and variation of the pH (Yu et al, 1993). After 25 min of reaction (Fig.…”

“…The metal content of the reconstituted Zn7-MT was confirmed using electrospray mass spectrometry (Yu et al, 1993). Metallothionein concentration was confirmed using the molar absorptivity of rabbit MT (48,200 mol" cm") at 220 nM (Vakik et al, 1987).…”