Metal- and Affinity-Specific Dual Labeling of Cysteine-Rich Proteins for Identification of Metal-Binding Sites

Abstract

Here, using human metallothionein (MT2) as an example, we describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity utilizing iodoacetamide (IAM) and N -ethylmaleimide reagents. We concluded that an S N 2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissociation. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addition reaction after separation by reverse-phase C18 at acidic pH. Finally, we evaluated the efficiency of the method by mapping metal-binding sites of Zn 7– x MT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resolution. The methodology presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-containing proteins.

Mass Spectrometry-Based Structural Analysis of Cysteine-Rich Metal-Binding Sites in Proteins with MetaOdysseus R Software

“…Interestingly, the y 31 fragment derived from backbone dissociation between α- and β-domain contained three Zn 2+ , probing the higher stability of Zn–S bonds and folding of the α-domain. 14 , 56 On the other hand, fragment b 17 with four Cys residues in the sequence contained one Zn 2+ . Analysis of the CID fragments from the data-independent acquisition (DIA) experiment showed that except the common b 17 fragment, most of them derived from the α-domain (C-terminus).…”

“…In our recent study, we attempted to develop an experimental approach that combined native MS and proteomics approaches to elucidate the metal–protein stoichiometry and topology of different Zn 7-x MT2 species. 2 , 14 Having prepared a metal–protein complex (e.g., Zn 4 MT2), the first step is to record mass spectra under nondenaturing conditions. The native-like structure is likely retained because of the kinetic trapping effect, which allowed us to determine the stoichiometry.…”

“… 2 , 12 − 16 Cysteine chemical labeling, improved ESI sources together with the recent advances in the transmission technology of Orbitrap and time-of-flight (TOF) mass analyzers have permitted the elucidation of the Cys oxidation state in the native-like structure. 2 − 6 , 14 , 17 The different reactivity between a free Cys residue and metal-binding Cys enables its identification with the use of an appropriate electrophile. 14 …”

“… 2 − 6 , 14 , 17 The different reactivity between a free Cys residue and metal-binding Cys enables its identification with the use of an appropriate electrophile. 14 …”

“…The characterization of metal–protein complexes, involving determination of the metal–protein stoichiometry, identification of metal-binding sites, or characterization of metal-coupled folding events, requires combining multiple MS-based approaches. 2 , 3 , 7 , 14 …”