Identification of the metal-binding intermediate of metallothioneins by ion mobility mass spectrometry (IM-MS): Evidence for cadmium(II) preferential binding in alpha-domain

摘要

The cadmium(II)-binding MT intermediates generated by either metallation of apoMT or demetallation of Cd_(u)MT were characterized and identified by ion mobility mass spectrometry. Addition of NEM either to block the free cystenines in partially-metallated MT or to substitute metals in Cd_(7)MT both resulted in stable four cadmium(II)-binding intermediates: Cd_(4)NEM_(9)MT and Cd_(4)NEM_(10)MT (in cases of NEM in large excess). IM-MS/MS experiments were performed to gain insight into alpha versus beta domain metallated sites. Fragments between different charge states or/and number of bound metal fall into different trendlines in IM-MS 2D plot, facilitating the identification of fragments with lower abundance and thereby increasing the coverage of fragmentation. The fragments of Cd_(4)NEM_(9)MT were matched to a fully NEM-labeled beta domain and cadmium(II)-saturated alpha domain. Proteolytic digestion showed that the metallated sites of four cadmium(II)-MT intermediates were cysteines in alpha domain and this cadmium(II)-saturated alpha domain remained intact, completely resistant to trypsin. Collectively these results suggest cooperative binding of Cd_(4)(alpha)MT, and the four cadmium(II) were tightly bound to alpha domain..