On-line coupling of continuous-flow gel electrophoresis with inductively coupled plasma-mass spectrometry to quantitatively evaluate intracellular metal binding properties of metallochaperones HpHypA and HpHspA in E. coli cells.

On-line coupling of gel electrophoresis with inductively coupled plasma-mass spectrometry (GE-ICP-MS) offers a strategy to monitor intracellular metals and their associated proteins simultaneously. Herein, we examine the feasibility of the GE-ICP-MS system in the quantitative analysis of intracellular metal binding properties using two Helicobacter pylori metallochaperones HypA and HspA overexpressed in E. coli cells as showcases. We show that parallel detection of metal and sulfur signals allows accurate quantification of intracellular metal-protein stoichiometries, even for metalloproteins that bind metal ions with micromolar affinities. Using this approach, we demonstrate that only a trace amount of Ni(2+) is associated with HpHypA in cells, distinct from the in vitro observation of stoichiometric binding, while HpHypA exhibits high fidelity towards its structural metal Zn(2+) with stoichiometric Zn(2+) binding. In contrast, HpHspA associates with Zn(2+), Ni(2+), Cu(2+) and Co(2+) from an essential metal pool with ca. 0.5 molar equivalents of total metals bound per HpHspA monomer. The metal binding properties of both HpHypA and HpHspA were altered by Bi(3+). The binding of both Zn(2+) and Ni(2+) to HpHypA was suppressed under the stress of Bi(3+) in cells, different from in vitro studies that showed that Bi(3+) interfered with Zn(2+) but not Ni(2+) binding. This study provides an analytical approach to investigate the intracellular metal selectivity of overexpressed metalloproteins.