Combining chemical labeling, bottom-up and top-down ion-mobility mass spectrometry to identify metal-binding sites of partially metalated metallothionein. Academic Article uri icon

abstract

  • Metalation and demetalation of human metallothionein-2A (MT) with Cd(2+) is investigated by using chemical labeling and "bottom-up" and "top-down" proteomics approaches. Both metalation and demetalation of MT-2A by Cd(2+) are shown to be domain specific and occur as two distinct processes. Metalation involves sequential addition of Cd(2+) to the -domain resulting in formation of an intermediate, Cd4MT. Chemical labeling with N-ethylmaleimide (NEM) and tandem mass spectrometry experiments clearly show that the four metal ions are located in the -domain. In the presence of excess Cd(2+), the Cd4MT intermediate reacts to add Cd(2+) to the -domain to yield the fully metalated Cd7MT. Demetalation occurs in the reverse order, i.e., Cd(2+) is removed (by EDTA) first from the -domain followed by Cd(2+) removal from the -domain. Metalation of human MT-2A is shown to be metal ion specific by comparing relative metal ion binding constants for Cd(2+) and Zn(2+).

published proceedings

  • Anal Chem

altmetric score

  • 0.25

author list (cited authors)

  • Chen, S., Russell, W. K., & Russell, D. H.

citation count

  • 35

complete list of authors

  • Chen, Shu-Hua||Russell, William K||Russell, David H

publication date

  • March 2013