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 SN2 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 Zn7–xMT 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.
Highlights
The Cys residue is a cellular target for reactive oxygen, nitrogen, and sulfur species, and it is posttranslationally modified in S-methylation and S-linked acylation, among other reactions.[13−16] Cys acts in multiple proteins as a redox switch, depending on the oxidative molecules and metal ion concentration.[17−20] Because of the aforementioned relevance, a range of experimental and theoretical tools has been developed aimed at identifying different Cys residue states in proteomes.[21−24] Most of the chemical tools are based on the nucleophilic reaction of Cys toward thiol-specific probes, which may exhibit different reactivity, enabling differentiation of the cysteine sulfur state.[25]
We demonstrated that IAM is more suitable than NEM to be used as the first labeling reagent in order to label free Cys residues
To identify the chemical or redox state of Cys residues in proteins, analytical methods are based on reactions with thiol-specific probes
Summary
The first part of this research attempted to study the kinetic and thermodynamic lability of Zn2+−thiolate bonds in fully saturated Zn7MT2 and to compare it with its cadmium counterpart. The number of Zn2+ detected by UV−vis practically did not change after addition of 40 equiv of alkylator, but the product ion [Zn4NEM9−11MT2]5+ showed up in the ESI-MS spectra [Figure 2A, Table 1, and Figure S4 (SI)] These results are consistent with the 10 Cys being NEM-labeled, suggesting a concomitant full β-domain modification, where 3 Zn2+ had been dissociated (Figure 2A). The complementary, double-labeling distribution obtained was centered, forming the IAM7NEM13MT2 species (8067.5 m/z) (Figure S16C, SI) This stoichiometry does not resemble that for coordination of a single ZnCys[4] site binding or a completely saturated α-cluster (Zn4S11) but rather suggests a structure with redistributed Zn2+ ions between domains.[31] a mixture of gas-phase ions with [Zn7−3IAMxMT2]5+ stoichiometry was annotated by nESI, which confirms the products IAM11NEM9MT2 (7817 m/z) and IAM3NEM17MT2 (8339 m/z) found in MALDI-MS (Figure S16B and Table S3, SI). The bottom-up MS results identified the tryptic fragment NEM1[21−30] with NEM-labeled Cys[21] as still existing in the spectra, confirming the gating role of Cys[21] for the seventh Zn2+ previously reported [Figure 1B and Figure S22 (SI)].31
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