Formation of oxidative and non‐oxidative dimers in metallothioneins: Implications for charge‐state analysis for structural determination

• Published in: Rapid communications in mass spectrometry Vol. 31; no. 24; pp. 2118 - 2124
• Main Authors: Irvine, Gordon W., Heinlein, Lina, Renaud, Justin B., Sumarah, Mark W., Stillman, Martin J.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 30.12.2017
• Subjects: Cysteine - analysis; Cysteine - chemistry; Cysteine - metabolism; Dimerization; Dimers; High resolution; Ionization; Ions; Mass spectrometers; Mass spectrometry; Metallothionein - analysis; Metallothionein - chemistry; Metallothionein - metabolism; Oxidation; Oxidation-Reduction; Protein Conformation; Proteins; Spectrometers; Spectrometry, Mass, Electrospray Ionization; Structural analysis; Zinc
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.8006

Rationale Metallothioneins (MTs) are a class of dynamic proteins that have been investigated extensively using mass spectrometric methods due to their amenability to ionization. Here we detect the formation of oxidative and non‐oxidative MT dimers using high‐resolution mass spectrometry (HRMS) which has previously been overlooked with lower‐resolution techniques. Methods Recombinant human MT1a and its isolated domain fragments were analyzed by high‐resolution Thermo Q‐Exactive and Bruker time‐of‐flight (TOF) mass spectrometers. Covalent Cys modification was performed using N‐ethylmalemide to probe the effect of Cys oxidation on dimer formation. Results Dimerization was detected in the analysis of select charge states of Zn7MT and apo‐βMT. Specifically, high resolution (140 k) revealed the +6 dimer peaks overlapping with the +3 charge state, but not with the other charge states (+4, +5, +6). The proteins with covalently modified Cys did not show dimer formation in any of their charge states. Apo‐α and apo‐βαMT also did not form dimers under the conditions tested. Conclusions Dimerization of MT was detected for zinc metalated and certain apo‐MT forms with HRMS, which was not seen with lower‐resolution techniques. These dimers appear overlapped only with certain charge states, confounding their analysis for structural characterization of MTs. The Zn‐MT dimers appeared to be non‐oxidative; however, the formation of dimers in the apo‐protein is likely dependent on Cys oxidation.