Chromatographic separation of similar post-translationally modified metallothioneins reveals the changing conformations of apo-MT upon cysteine alkylation by high resolution LC-ESI-MS.

Metallothioneins (MTs) are a class of small cysteine-rich proteins essential for Zn and Cu homeostasis, heavy metal detoxification, and cellular redox chemistry. Herein, we describe the separation and characterization of MTs differentially modified with N-ethylmaleimide (NEM) by liquid chromatography-mass spectrometry (LC-MS). The full-length recombinant MT isoform 1a as well as is isolated domain fragments were first alkylated, then separated on column with subsequent detection by ultra-high resolution ESI-MS. Different behavior was observed for the three peptides with the full-length protein and the isolated α-domain exhibiting similar separation characteristics. For the isolated β-domain, the smallest peptide with 9 cysteines in the sequence, each alkylated species was well separated, indicating large changes in protein conformation. For the full-length (20 cysteines in the sequence) and α-domain (11 cysteiens in the sequence) peptides, the apo- and lightly alkylated species co-eluted, indicating similar structural properties. However, the more extensively alkylated species were well separated from each other, indicating the sequential unfolding of the apo-MT peptides and providing evidence for the mechanistic explanation for the cooperative alkylation reaction observed for NEM and other bulky and hydrophobic alkylation reagents. We show for the first time clear separation of highly similar MTs, differing by only +125 Da, and can infer structural properties from the LC-MS data, analogous to more complicated and less ubiquitous ion-mobility experiments. The data suggest a compact globular structure for each of the apo-MTs, but where the β-domain is more easily unfolded. This differential folding stability may have biological implications in terms of domain-specific participation of MT in cellular redox chemistry and resulting metal release.