title: Combining chemical labeling, bottom-up and top-down ion-mobility mass spectrometry to identify metal-binding sites of partially metalated metallothionein.
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 α-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 α-domain. In the presence of excess Cd(2+), the Cd4MT intermediate reacts to add Cd(2+) to the β-domain to yield the fully metalated Cd7MT. Demetalation occurs in the reverse order, i.e., Cd(2+) is removed (by EDTA) first from the β-domain followed by Cd(2+) removal from the α-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+).
authors: Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
authors: Shu-Hua
authors: SH
authors: Chen
authors: William K
authors: WK
authors: Russell
authors: David H
authors: DH
authors: Russell
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D007553:Isotope Labeling / Q000379:methods; D008668:Metallothionein / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C491053:MT2A protein, human; D008668:Metallothionein